Marble Burying and Nestlet Shredding as Tests of Repetitive, Compulsive-like Behaviors in Mice

A primary advantage of the marble burying and nestlet shredding tests is the fact that each depends on natural and spontaneous behaviors of mice that can be quantified after test completion, allowing a single investigator to set up an experiment and then remain outside of the testing room until sessions end. A critical component of the marble burying test is the careful removal of the subject from the cage after testing. Mice typically attempt to avoid capture and, in the process, can dislodge or move marbles that were partially buried or partially bury marbles that were not buried at the end of the test session. Figure 1A shows a test cage just prior to introduction of a subject, as viewed from above. We prefer a precise alignment of marbles in this fashion which can subsequently be used to track movement of the marbles, in addition to burying (if desired). In an illustrative experiment, we compared marble burying by wild type (WT) mice to mice lacking the gene for tryptophan hydroxylase 2 (TPH2), the initial and rate limiting enzyme in the biosynthesis of brain serotonin (5HT). As a result of the null mutation in the gene for TPH2, these mice have no measureable brain 5HT^15,16,18. These mice show a very interesting behavioral phenotype characterized primarily by repetitive, compulsive-like digging and burying^15,16. Figure 1B shows a representative cage from WT mice and Figure 1C is a cage from a TPH2 knockout (KO) mouse after burying. The cage of the WT mouse is typical and shows few marbles buried (N = 3 in this example) and little displacement from their original locations. On the other hand, the TPH2 KO cage shows significant numbers of buried marbles (N = 17 in this example; unburied marbles marked with arrows) and an extensive displacement of marbles from their initial locations. The appearance of the bedding surface is also indicative of burying and digging behavior. Figure 1D, as viewed along the surface of the bedding, shows a level bedding surface prior to testing. Figure 1E shows that bedding also remains relatively undisturbed by a WT mouse. In contrast, Figure 1F shows a typical cage from a TPH2 KO mouse after the test session with a very undulating surface and deep troughs and peaks throughout the cage. Figure 2 (reprinted from Angoa-Pérez et al.¹⁵ with permission from Wiley) illustrates the principal result of this test which is simply the number of marbles buried. Clearly, the TPH2 KO mice buried significantly more marbles than their WT counterparts (13 versus 4 out of 20, respectively). Perhaps the one challenge in scoring this test is the definition of a buried marble. In this test, a marble is scored as buried if two-thirds of its surface is covered by bedding¹⁰. WT mice bury few marbles, so scoring them is generally not a problem. However, we find that TPH2 KO mice not only bury marbles but move large amounts of bedding around the cage as they burrow and dig, making scoring a greater challenge. Because this scoring is somewhat subjective, we use 2-3 independent scorers who are blind to the genotype or treatment of the test mice. As long as marbles are not accidentally displaced when test subjects are removed from the cage, the marble burying test is a robust and sensitive method for the analysis of repetitive, compulsive-like behaviors in normal mice and in mice that have been genetically modified. The usefulness of this test for assessing the effects of numerous classes of drugs on repetitive behaviors has also been established^6,7,10.

Nestlet shredding is another test that takes advantage of the natural proclivity of mice to shred practically any material for nest construction. Males and female mice are both reliable shredders. As in the marble burying test, a single investigator can set up large numbers of test cages and then leave the test room for the duration of the test session. Figures 3A and 3B show two cages from WT mice after conclusion of the test. The nestlets in Figures 3A and 3B were 11% and 27% shredded, respectively. Figures 3C and 3D show representative nestlets from two TPH2 KO mice and show that nestlets are much more extensively shredded (i.e. 51% and 100% shredded in Figures 3C and 3D, respectively). Because nestlets are carefully weighted before placement into the cage, the analytical task in this test is to weigh the remainder of the intact nestlet after test completion to quantify the amount of nestlet shredded. In our experience, two issues can slightly complicate weighing of the nestlets after shredding. First, if the nestlets are even slightly moist, the weight of the remaining nestlet will be artificially high. To avoid this situation, we allow shredded nestlets to dry O/N to remove any moisture and we withhold food and water bottles (as sources of moisture) from cages during the test. Second, the shredded nestlets are not always as "cleanly" or totally shredded as in Figure 3D. It can be seen that some nestlets contain adherent material that has clearly been shredded from the main body of the nestlet, but is still clinging to the intact part. As in the marble burying test, we use 2-3 raters to judge the amount of adherent but shredded material that should not be considered part of the remaining intact nestlet. This material is then carefully teased away from the intact nestlet with forceps and the remaining unshredded portion is weighed. An example of this condition is presented in the inset to Figure 3A with arrows pointing to material that should be removed prior to weighing or the remaining nestlet. Figure 4 (reprinted from Angoa-Pérez et al.¹⁵ with permission from Wiley) illustrates a representative outcome of nestlet shredding when comparing WT mice to TPH2 KO mice. It can be seen that TPH2 KO mice show significantly more shredding than WT controls (~ 50% versus 10% shredded, respectfully). The nestlet shredding test is a simple yet robust and sensitive method for assaying repetitive, compulsive-like behaviors in mice. Nestlet shredding is also a good compliment to the marble burying test and its effectiveness in testing a wide variety of drugs for their effects on repetitive behaviors has been well established¹⁰.

Figure 1. These photographs show representative cages before and after the marble burying test using WT and TPH2 KO mice as subjects. Panels A-C are photographed from above the cages and panels D-F are photographed along the surface of each cage. A) An example of a typical cage prior to introduction of a test subject, showing initial marble alignment; B) An example of a representative cage from a WT mouse after burying. In this example, few marbles are buried (N = 3) or displaced from their initial location; C) An example of a typical cage after testing a TPH2 KO mouse showing that 17 of the 20 marbles were buried in this case. Unburied marbles are indicated with arrows; D) A view of the bedding surface before introduction of a test mouse; E) An example of alterations in the bedding surface after removal of the WT mouse and F) An example of the bedding surface after testing a TPH2 KO mouse. The photograph in panel F cannot fully demonstrate in two-dimensions the disruption of the bedding material caused by the TPH2 KO mouse. Not only have most marbles been buried, the surface of the bedding appears to have been excavated by the TPH2 KO mouse. Areas of large troughs and peaks are clearly visible and it is typically the case that mice will dig and displace all bedding from one corner of the cage. Click here to view larger image.

Figure 2. This graph shows typical performance of WT and TPH2 KO mice on the marble burying test. Observers blind to the genotype of test mice score each cage for the number of marbles buried (i.e. two-thirds of their surface covered with bedding). The scores for each test subject are averaged for all scorers and plotted as number of marbles buried versus mouse genotype. TPH2 KO mice (N= 20; 10 males and 10 females) bury significantly (two-way ANOVA, F_1,66 = 35.61, p <0.05; comparing sex and genotype) more marbles than WT mice (N= 37; 26 males and 11 females). Males and females did not differ in marble burying so data was combined across this variable within each genotype. Results are mean ± standard error of the mean. *** indicates p <0.001 for Bonferroni post hoc comparisons between WT and KO mice. This figure is reprinted from Angoa-Pérez et al.¹⁵ with the permission of Wiley. Click here to view larger image.

Figure 3. These photographs show representative nestlets after shredding. A) and B) Examples of nestlets from 2 male WT mice showing minor amounts of shredding (i.e. 11% and 27% shredded in A and B, respectively, in these examples); C) and D) Examples of nestlets from 2 male TPH2 KO mice showing greater amounts of shredding (51% and 100% shredded in C and D, respectively, in these examples). Note in some cases that shredded material remains adherent to the main body of the unshredded nestlet (see inset to panel A). We use 2-3 scorers to judge the amount of shredded material that can be removed from the unshredded remainder of the nestlet. After this material is removed carefully, the remaining unshredded nestlet is weighed. Click here to view larger image.

Figure 4. This graph shows typical performance of WT and TPH2 KO mice on the nestlet shredding test. The weight of the remaining unshredded nestlet is divided by its initial weight to calculate the percentage of nestlet shredded and plotted versus mouse genotype. The TPH2 KO mice (N = 6 male mice) shred significantly (Student's t-test, t₁₀ = 3.07, p <0.05) more nestlet material than WT mice (N = 6 male mice). Results are mean ± standard error of the mean. * indicates p <0.05. This figure is reprinted from Angoa-Pérez et al.¹⁵ with the permission of Wiley. Click here to view larger image.