Digging behavior discrimination test to probe burrowing and exploratory digging in male and female mice

Abstract

Digging behavior is often used to test motor function and repetitive behaviors in mice. Different digging paradigms have been developed for behaviors related to anxiety and compulsion in mouse lines generated to recapitulate genetic mutations leading to psychiatric and neurological disorders. However, the interpretation of these tests has been confounded by the difficulty of determining the motivation behind digging in mice. Digging is a naturalistic mouse behavior that can be focused toward different goals, that is foraging for food, burrowing for shelter, burying objects, or even for recreation as has been shown for dogs, ferrets, and human children. However, the interpretation of results from current testing protocols assumes the motivation behind the behavior often concluding that increased digging is a repetitive or compulsive behavior. We asked whether providing a choice between different types of digging activities would increase sensitivity to assess digging motivation. Here, we present a test to distinguish between burrowing and exploratory digging in mice. We found that mice prefer burrowing when the option is available. When food restriction was used to promote a switch from burrowing to exploration, males readily switched from burrowing to digging outside, while females did not. In addition, when we tested a model of intellectual disability and autism spectrum disorder that had shown inconsistent results in the marble burying test, the Cc2d1a conditional knockout mouse, we found greatly reduced burrowing only in males. Our findings indicate that digging is a nuanced motivated behavior and suggest that male and female rodents may perform it differently.

Keywords: RRID:IMSR_CRL:27; RRID:IMSR_JAX:005359; RRID:IMSR_TAC:b6; RRID:MGI:5449582; behavioral analysis; burrowing; digging; repetitive behaviors.

Figure 1.. Stability of performance on Digging Behavior Discrimination test.

Three cohorts of mice (M1: N=11; M2 N=13; F: N=10) were tested independently to assess stability of burrowing and digging performance and define possible sex differences. (A) Test chamber set up and schematic of digging and burrowing zones. (B-C) Males (M1, M2) covered similar distances at equal speed, while females showed increased motor activity (B) and speed (C). (D–F.) Different cohorts showed mostly consistent digging performance inside and outside the burrow area. Occupancy in the burrow area (D.), total time spent digging in both areas (E.) and outside (F.) were similar. (G.) Most of the digging time was spent burrowing. (H-I.) Latency to burrow and time to empty the burrow were also similar in all cohorts. (J.) Time spent in direct interaction with substrate in the burrow was variable with females significantly lower from the M1 group, but not M2. (K.) The burrowing rates, grams of burrow substrate removed per minute, were not significantly different, but M1 trended towards a slower rate. (L.) Overall, most animals efficiently removed the burrowing substrate from the tube by the end of the test. (M.) The substrate was then distributed over the area of the cage (example of spread soft bedding outlined in red on the right). Values are presented as means ± SEM. Symbols are individual mouse data points. *p < 0.05, **<0.01, or ***<0.001 following multiple comparison tests. All unmarked comparisons were not significant. Additional statistical information is reported in Suppl. Table 1.

Figure 2.. Male and female mice show different free digging and burrowing performance after a food restriction challenge.

(A.) Two cohorts of mice (Male: N = 10; Female: N = 7) were assessed using the DBD test at baseline (Base), during food restriction (FR), and once recovered from food restriction (Rec). (B – C.) Female mice covered more distance (B.) at a faster pace (C.) than male counterparts at baseline and showed a more prominent drop to male-like levels of activity during FR. Both sexes recovered to baseline levels. (D.) Total digging activity remained similar. (E.) Males spent significantly more time digging during FR, while females maintained constant digging performance. (F-G.) Latency to burrow and burrowing rate did not change significantly during FR or recovery. (H-I.) Analysis of the ratio between burrowing and total digging and percentage of material removed from the burrow revealed differences in response between males and females. Females engage in limited burrowing at baseline and FR, but increase during recovery, whereas males burrow substantially at baseline, reduce during FR, and return to baseline performance during recovery. Values are means ± SEM. Symbols are individual mouse data points. *p < 0.05, **<0.01, or ***<0.001 following multiple comparison tests. All unmarked comparisons were not significant. Additional statistical information is reported in Suppl. Table 2.

Figure 3.. Plasma Corticosterone levels at baseline and with food restriction.

(A) Female mice exhibited higher concentrations of CORT than males under baseline conditions. (B) CORT levels increased in both sexes during FR, but only females retained high levels once recovered from FR. Values are means ± SEM. Symbols are individual mouse data points. *p < 0.05, **<0.01, or ***<0.001

Figure 4.. Cc2d1a cKO males show reduced burrowing performance.

4 cohorts of mice (Cont M: N=8; cKO M: N=8; Cont F: N=10; cKO F: N=10) were tested independently to assess the protocol sensitivity to an animal model of ASD. (A-B.) Between genotype male and females covered similar distances at equal speed, while females showed increased motor activity (A.) and speed (B.) when compared to males. (C.) Both control and cKO females showed significantly reduced total digging behavior when compared to males. (D.) cKO males spent significantly more time digging than both female cohorts and showed a trend towards more digging compared to control males. (E.) All animals spent more time burrowing than digging outside, though male cKOs and females showed great variability. (F.) cKO males burrowed significantly less material than wild type males, a difference not seen between female genotypes. Values are means ± SEM. Symbols are individual mouse data points. *p < 0.05, **p<0.01, or ***p<0.001 following multiple comparison tests. All unmarked comparisons were not significant. Additional statistical information is reported in Suppl. Table 4.