Experiment 1: strain distribution pattern in MB, OFA, and LD
The data in clearly demonstrate the significant differences in marble burying across the ten inbred strains of mice, with a significant main effect of strain [F(9,110)= 9.13, p<0.001]. Strains AKR/J, FVB/NJ, and CBA/J buried the most marbles while 129S6/SvEv Tac mice buried the least.
Fig. 1 Behavioral responses of ten inbred strains of mice on the marble-burying, open-field, and light–dark tests. The number of marbles buried among inbred strains of mice (a); open-field exploratory activity as measured by the total distance traveled (more ...)
illustrates the strain distribution for the total distance, center ratio, and “stereotypy” measures in the open-field test and the number of light–dark transitions in the LD test. In order to easily compare the strain distribution pattern, the order of strains in all figures for match the order from the marble-burying test (see ). Significant main effects of strain were found for each of the OFA and LD measures [total distance: F
<0.001; center ratio: F
<0.001; “stereotypy”: F
(9,110)= 2.72, p
=0.007; LD transitions: F
<0.001]. It is important to note that the OFA and LD strain survey data presented here are consistent with strain survey data in the literature (Bolivar et al. 2000
; Bouwknecht and Paylor 2002
; Milner and Crabbe 2008
Pearson’s correlation analysis () revealed that the anxiety-like measures for LD (transitions) and OFA (center ratio) significantly correlated with one another (R= 0.826, p=0.003), but neither LD nor OFA measures correlated with the number of marbles buried (R=0.434, p=0.211; R=0.505, p=0.137, respectively). In addition, activity in the OFA (total distance traveled) significantly correlated with LD transitions (R=0.852, p=0.002) and the OFA center ratio (R=0.748, p=0.013), but not with the number of marbles buried (R=0.531, p=0.115). Interestingly, although it failed to reach statistical significance, the measure with the highest correlation value with the number of marbles buried was the “stereotypy” measure from OFA (R=0.620, p=0.056).
Table 1 Pearson r correlations between measures within three behavioral assays: marble burying (# marbles buried), light–dark exploration (transitions), and open-field activity (total distance traveled, center/total distance traveled ratio, “stereotypy”) (more ...)
Experiment 2a: reducing the novelty of marbles by exposure in home cage
In the home cage habituation paradigm, mice were exposed to marbles in their home cage for either 1 or 5 days, and their performance in the marble-burying experiment was compared to mice not preexposed to marbles. There were no significant differences in the number of marbles buried between the three testing groups (F(2,21)=0.17, p=0.847; ).
Fig. 2 The number of marbles buried in mice naïve (standard) or familiarized (1 and 5 day) to marbles in the home cage prior to testing (a); the number of marbles buried following repeated testing during five consecutive days (b) or five 1-h intervals (more ...)
Experiment 2b: reducing the novelty of marbles by repeat testing
In this experiment, mice repeatedly went through the marble-burying test in order to reduce marble novelty. When mice were tested once daily for five consecutive days, we observed no difference in the number of marbles buried over the 5-day testing period (F(4,44)=2.26, p= 0.078; ). However, mice tested five times within the same day did exhibit a significant main effect of test number (F(4,44)=4.15, p=0.006; ). Follow-up analysis using within-subject contrasts revealed a significant decrease in marbles buried during the fifth test compared to the first test [F(1,11)=4.95, p=0.048], while neither the second, third nor fourth test significantly varied from the first test.
Experiment 2c: marbles vs. food
To determine if mice will bury a very familiar object, such as food pellets from their own cage, we compared the burying behavior of mice presented with either marbles or food. It is important to note the difficulty in directly comparing marbles to food since they are different in size and shape. Each mouse was tested once with marbles and once with food pellets using a balanced crossover design. The results illustrate that mice clearly bury familiar food pellets, although the number buried is less than the number of marbles buried (F(1,20)=4.86, p=0.039; ). ANOVA revealed no effect of test history [F(1,20)=0.09, p=0.772], indicating that the results are not significantly influenced by which test was performed first.
The number of marbles or food pellets buried. All values represent means ± standard error of the mean. *p<0.05
Experiment 3: do mice avoid marbles when given an opportunity?
In order to determine if mice will avoid marbles when given the opportunity, mice were tested for marble-burying behavior in one of two conditions: a large cage with marbles spread throughout the entire bedding surface or a large cage with marbles covering only half the surface. No significant difference was found between either of the two cage conditions (F(1,18)=0.05, p=0.831; ) indicating that mice bury similar amounts of marbles even when provided sufficient space to “avoid” the marbles.
The number of marbles buried in a large arena containing marbles spread across either the entire arena (full) or only half the arena (half). All values represent means ± standard error of the mean
Experiment 4a: digging in marble vs. nonmarble cage
We evaluated mouse-digging behavior in cages with clean bedding in the presence or absence of marbles to determine if marbles affect digging behavior. clearly demonstrates that the presence of marbles did not influence the number of digging observations recorded [F(1,24)= 0.11, p=0.739].
Fig. 5 Digging behavior in mice. The number of digging observations in either the presence or absence of marbles (a); the number of digging observations in the presence of either marbles or food pellets (b). All values represent means ± standard error (more ...)
Experiment 4b: digging in the presence of marbles vs. food
When mice are placed in a cage with either marbles or food pellets present on the bedding surface, we find that the novelty of an object (marble) has no affect on the number of digging observations observed when compared to digging in the presence of a more familiar object (food pellet) (F(1,22)=0.06, p=0.808; ).
Experiment 4c: will mice selectively dig in a location where marbles are present or absent?
To determine if digging behavior changes across repeated testing, we evaluated burying and digging behavior in a large cage containing marbles on half of the surface of the bedding. Consistent with the data from , the number of marbles buried did not significantly change across testing days (F(4,28)=1.92, p=0.135; ). Furthermore, digging observation on the marble side also did not significantly change over the multiple testing days (F(4,28)=1.32, p= 0.288; ). On the first day of testing, mice spent approximately 50% of the time on the marble side of the cage, and while repeat testing led to a decrease in the percentage of time spent on the marble side, this was not significant [F(4,28)=2.35, p=0.079; data not shown].
Fig. 6 Digging activity and marble burying in a large arena half covered with marbles across five testing days. The total number of marbles buried and the number of digging observations on the marble side of the arena (a); the % of time spent digging on each (more ...)
When examining the percentage of digging events observed for each of the two sides, an overall main effect of testing day was observed (F(4,56)=3.25, p=0.018; ); however, no interaction was observed between testing day and cage side [marble vs. nonmarble; F(4,56)= 0.84, p=0.509]. In order to compare the number of marbles buried to digging activity on the marble side, we calculated a ratio for % marbles buried: % digging events observed on the marble side (). Repeated-measures ANOVA demonstrated a significant main effect of day [F(4,24)= 3.68, p=0.018], and follow-up analysis illustrated a significant increase between test days 1 and 5 [F(1,6)= 9.11, p=0.023] indicating that there is some dissociation between marble burying and digging and that they are not completely coupled responses.
Pearson’s correlation was implemented in order to evaluate how marble burying in this experimental design might correlate with digging and, more specifically, the location of those digging events (marble vs. nonmarble side). For each of the five testing days, the number of marbles buried significantly correlated with both the total number of digging events as well as the digging events observed on the marble side (p<0.05; ). On the other hand, marble burying significantly correlated with digging observed on the nonmarble side only on days 1 and 2.
Pearson r correlations between the total number of marbles buried and total digging events observed, digging events observed on the marble side, or those observed on the nonmarble side
Experiment 5: reducing the novelty of the bedding
To determine the impact of novel “burying media”, we measured marble-burying and digging behavior in a cage with new bedding (i.e., traditional marble-burying test setup) compared to burying in a cage using “home cage” bedding. displays that test media can impact marble-burying and digging responses in a complex manner that is dependent on familiarity.
Fig. 7 The number of marbles buried (a) or digging observations (b) in either the home cage or a cage with new bedding across two tests. Asterisk, single number sign, double number sign, and dollar sign indicate significant differences (p<0.05). The (more ...)
For marble burying, there was an overall order effect on marble burying [test 2 > test 1; F(1,20)=9.10, p=0.007], but the main effect of test media was not significantly different [F(1,20)=3.91, p=0.062]. However, the interaction (test media × test order) was significant [F(1,20)=4.77, p= 0.041]. Simple effects analysis of the interaction indicated that there was a significant effect of test order, but only in the home testing condition [test 2 > test 1; F(1,10)=7.54, p= 0.021]. There was no significant order effect in the traditional new cage setup [F(1,10)=1.68, p=0.224]. In addition, the simple effects analysis also indicated that mice in the home cage buried fewer marbles compared to the new cage, but this effect was only observed during test 1 [F (1,10)=5.86, p=0.036]. Thus, the test media can alter marble burying, but only in naive mice.
Similar to that observed for marble burying, there was an overall effect of test order for the digging measure [test 2 > test 1; F(1,20)=4.73, p=0.042], but the main effect of test media was not significantly different [F(1,20)=0.35, p= 0.560]. However, the interaction (test media × test order) was significant [F(1,20)=10.86, p=0.004]. Simple effects analysis of the interaction indicated that there was a significant effect of test order, but only in the home testing condition [F(1,10)=11.56, p=0.007]. There was no significant test order effect in the traditional new cage setup [F (1,10)=0.89, p=0.368]. In contrast to the marble-burying findings, the simple effects analysis also indicated that mice in the home cage had fewer digging events compared to those in the new cage during test 1 [F(1,10)=6.23, p= 0.032], but significantly more during test 2 [F(1,10)=5.34, p=0.043]. Thus, digging behavior is also affected by the familiarity of the test media in opposite directions dependent on prior exposure to the marble test.