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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-Prez 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 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.

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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

With respect to existing methods (e.g. genetic, pharmacological, and neurodevelopment models) the marble burying and nestlet shredding tests have numerous advantages. Both are readily available to most labs without the need for sophisticated and expensive equipment. These tests are simple to apply, they can be formatted for high-throughput analyses and they are easy to score reliably. With slight modifications, these models can provide additional valuable information that can be useful when studying more complex behaviors and disorders. For instance, test sessions in both models can be video-taped and mice can be scored for locomotor activity, distance traveled, time spent moving, and percent of time spent in each region of the test cage (e.g. center versus perimeter). Measures such as these can provide revealing information on levels of anxiety, particularly if using subject mice with genetic manipulations that might be anxiogenic. Drug treatments can also modify repetitive behaviors indirectly by altering locomotor activity, so the parallel measure of this parameter along with marble burying or nestlet shredding could reveal drug-induced effects on movement.

One limitation of the marble burying test relates to the question of whether marble burying actually represents compulsive-like behavior or if the marbles evoke novelty-induced or anxiety-like responses in mice. Careful study has shown that the glass marbles do not serve as a fear- or anxiety-producing stimulus19,20 and attempts to habituate mice to the marbles do not change burying. It could also be argued that marble burying is actually secondary to digging and burrowing in rodents. Therefore, mice do not bury marbles specifically but in the process of digging and burrowing, marbles become covered by the bedding. This potential limitation can be tempered by the observation that digging correlates with burying and not with other behaviors emitted during this test19-21. Another limitation of marble burying is under-estimation of the behavior in animals that show excessive amounts of digging and burrowing (e.g. TPH2 KO mice). These mice dig intensely in the absence of any objects in the test cage16 and a similar situation could occur in other genetically modified mice or in subjects treated with certain drugs. Thus, it is likely that marbles are buried and subsequently uncovered, to be scored inaccurately as unburied. Finally, a limitation that applies to both the marble burying and nestlet shredding tests is the fact that mice will often climb on and hang from the wire cage tops. This competing behavior will diminish the amount of time spent in marble burying and nestlet shredding. This climbing behavior is natural in rodents and could represent a repetitive behavior as well. However, to avoid this potential problem, flat wire tops or filter-top covers can be used.

A critical step for both protocols is the use of fresh bedding to minimize the influence of interfering olfactory cues from subject to subject. Marbles should be cleaned carefully before each test and marbles and nestlets should always be handled with gloves for the same reason. To the extent possible, flat wire cage covers or filter-top covers should be used with deeper cages so that mice cannot reach the wire covers and spend time climbing. In addition, cages should have a minimum size of 26 cm x 48 cm x 20 cm to allow even dispersion of the marbles so that mice can ambulate in the cage without stepping on the marbles. At the completion of marble burying sessions, mice must be removed carefully to avoid movement of the marbles prior to scoring. After the completion of the nestlet shredding test, it is critical to allow nestlets to dry before their post-session weights are measured to avoid under-estimation of shredding. Finally, both tests should be scored by observers blinded to any variables or treatment applied to the subjects under study (e.g. genotype of mice being compared, drug treatment).

A. Nestlets after the 30 min-observation period are documented for all tested animals (n = 9 per genotype). Numbers inside the panels indicate the test ID (black: wild type; red: Gnpat KO). Note the markedly higher shredding activity of wild type compared with Gnpat KO mice. B. The difference in nestlet weight before and after the trial was calculated for each test animal and results are shown as individual data points. Grey bars indicate the mean value. Numbers next to the data points designate the mouse test ID, as defined in (A). Statistical testing was performed using Mann-Whitney U-test (**p < 0.01). The quantitative data shown here have also been presented in our previous publication (Dorninger et al., 2019a).

Both of the presented assays have several obvious advantages: They are low-cost experiments, by which a significant amount of data can be obtained in a relatively short time frame. Furthermore, they can be reliably performed by investigators with only modest experience in behavioral analysis. Both tests are hardly dependent on physical abilities of the animals, thus allowing the use of genetically modified mouse models with marked phenotypes. For example, we used mice with considerable developmental defects and visual impairments (the ether lipid-deficient Gnpat knockout (KO) mouse model) (Rodemer et al., 2003), which precluded the application of many behavioral tests. However, as demonstrated by their performance in the nest building assay (Dorninger et al., 2019a), the animals are perfectly capable to utilize nestlets. Certainly, the simplicity of the assays also comes with some caveats: Both tests provide only an initial estimation on potential features of neurodevelopmental disorders and should not be used isolated. Also, they do not yield numeric data that can be directly related to the extent of human disease. Instead, they need to be complemented by additional behavioral tests assessing similar features (Silverman et al., 2010) and, optimally, embedded in a battery of assays allowing a comprehensive description of the behavioral phenotype. Furthermore, results need to be interpreted with care: Excessive nestlet shredding is generally seen as an indicator of repetitive behavior, a symptom also seen in human autism spectrum disorders or obsessive-compulsive disorder (Angoa-Perez et al., 2013). On the contrary, reduced engagement in the task may be viewed as restricted interest in novel objects, another feature commonly associated with autism (Bernard et al., 2015; Dorninger et al., 2019a). We feel that both interpretations are valid but need to be considered in the context of other aspects of behavior of a certain mouse model. Nest building, in turn, can be indicative of general well-being, distress or pain (Jirkof, 2014). At the same time, impairments in this task could hint towards structural or functional deficits in the brain, as demonstrated by mouse models of brain injury or with genetic modifications (Lijam et al., 1997; Deacon et al., 2002; Sager et al., 2010). 2351a5e196