All tests were performed with adult male mice 8 - 12 weeks of age that were either homozygous for the Ube3a null allele (Ube3am-/p-, n = 11) or inherited the null allele through the maternal germline (Ube3am-/p+, n = 25 except for measurements of area covered where n = 23). Wild-type littermates (Ube3am+/p+, n = 36) of these animals were used as controls. The construction and breeding of the Ube3a deficient mice have been described by Jiang and colleagues [11].
The results section is divided into two segments: first, we report results from analyzing the entire length of the path, covering all of the arena (gray area in Figure 1A) under control conditions (with the two small cages empty) and under test conditions (with a stranger mouse present in one of the cages). Second, we describe results from analyzing the behavior directly in front of the two small cages, differentiating between the empty cage and the cage with the stranger mouse. Here, pathway analysis was limited to within the two small zones shown in gray in Figure 1B.
Whole Arena Measurements
Representative examples of exploratory behavior of Ube3a deficient and wild-type mice under control and test conditions (without and with a stranger mouse present, respectively) are visualized for qualitative comparison in Figure 2 as movement path vs. time plots (Figure 2A-C, J-L), and color maps for duration (Figure 2D-F, M-O) and movement speed (Figure 2G-I, P-R). Quantitative comparisons are shown in subsequent figures.
Movement path vs. time plots show the exploratory paths overlying the video image of the arena with time plotted along the z-axis (Figure 2A-C, J-L). Visual comparison of the path vs. time plots shows that both Ube3a deficient mice had shorter exploratory paths and covered less area in their exploration than wild-type mice under both control (Figure 2A-C) and test conditions (Figure 2J-L). The quantitative comparison of path length under control and test conditions revealed that Ube3am-/p- and Ube3am-/p+ mice had significantly shorter exploratory paths than their wild-type litter mates, under both control and test conditions (Figure 3A) (F [5,138] = 33.29, p ≤ 0.001, Tukey post hoc (TPH) results for pairwise comparison to wild type litter mates under control conditions: Ube3am-/p-, p ≤ 0.001, mdiff = -1690.23, serr = 242.46 and Ube3am-/p+, p ≤ 0.001, mdiff = -1455.57, serr = 183.22; TPH results for pairwise comparison to wild type litter mates under test conditions: Ube3am-/p-, p ≤ 0.001, mdiff = -1223.17, serr = 242.46; Ube3am-/p+, p ≤ 0.001, mdiff = -1061.58, serr = 183.22). Similarly, quantitative comparison of the area covered by the paths (Figure 3B) showed that wild-type mice covered a significantly larger proportion of the arena than Ube3a deficient mice (F [5,134] = 20.89, p ≤ 0.005, TPH. results for pairwise comparison to wild type litter mates under control conditions: Ube3am-/p-, p ≤ 0.002, mdiff = -8.16, serr = 2.07; Ube3am-/p+, p ≤ 0.005, mdiff = -5.81, serr = 1.6; TPH results for pairwise comparison to wild type litter mates under test conditions: Ube3am-/p+, p ≤ 0.001, mdiff = -8.22, serr = 1.6; Ube3am-/p-, p ≤ 0.001, mdiff = -9.01, serr = 2.07). There were no significant differences in path length or area covered between Ube3am-/p- and Ube3am-/p+ mice under either condition.
Color maps for duration of stay (Figure 2D-F, M-O) show sites were mice lingered for more than 60 seconds as red and purple spots (two examples marked by arrows in Figure 2D). Both Ube3am-/p- and Ube3am-/p+ mice (Figure 2D-F) lingered frequently for 60 seconds or more at various sites distributed across most of the two side chambers with a preference of lingering close to a wall (Figure 2D,E). Mice rarely lingered in the middle chamber.
By comparison, the wild-type mouse had overall fewer places where it lingered for more than 60 seconds and lingering sites were concentrated in front of the two empty cages (Figure 2F). A quantitative comparison of lingering behavior as the proportion of time the mice spent moving during the 10 min trials revealed that wild-type mice spent significantly more time moving than did Ube3a deficient mice (F [5,138] = 30.99; p ≤ 0.001; TPH results for pairwise comparison to wild type litter mates under control conditions: Ube3am-/p-, p ≤ 0.001, mdiff = -13.77 serr = 2.33; Ube3am-/p+, p ≤ 0.001, mdiff = -11.62, serr = 1.76, TPH results for pairwise comparison to wild type litter mates under test conditions: Ube3am-/p-, p ≤ 0.001, mdiff = -10.02, serr = 2.33; Ube3am-/p+, p ≤ 0.001, mdiff = -9.33, serr = 1.76). Comparison of the color maps for speed (Figure 2 G-I, P-R) show that Ube3a deficient mice generally moved at a slower speed than their wild-type littermates. Quantitative comparison revealed significantly lower movement speeds in both Ube3a deficient genotypes compared to wild-type littermates (F [5,138] = 32.52, p ≤ 0.001; TPH results for pairwise comparison to wild type litter mates under control conditions: Ube3am-/p-, p ≤ 0.001, mdiff = -2.83, serr = 0.41; Ube3am-/p+, p ≤ 0.001, mdiff = -2.47, serr = 0.31; TPH results for pairwise comparison to wild type litter mates under control conditions: Ube3am-/p-, p ≤ 0.001, mdiff = -2.07; serr = 0.41; Ube3am-/p+, p ≤ 0.001, mdiff = -1.81, serr = 0.31) (Figure 3D). This difference was independent of social context, i.e. of the presence or absence of a stranger mouse. There was no significant difference in movement speed between Ube3am-/p- and Ube3am-/p+ mice under either condition.
During the test trials (with a stranger mouse present) mice from all three genotypes spent extended amounts of time in front of the cage with the stranger mouse (cage marked "S" in Figure 2M-R) signified by the accumulation of lingering sites (red/purple dots). This was interpreted as social interest in the stranger mouse and was quantified as the total amount of time the mice spent inside the area in front of the cage. We also measured the number of visits or entries into the area in front of the stranger's cage. A detailed description of results for the behaviors directly in front of cages is given below.
We consistently observed that mice of all three genotypes moved slower, had shorter track lengths and spent less time moving during the second trial, i.e. when the stranger mouse was present in one of the cages. The decreases in these variables were comparable in magnitude between genotypes but significant differences for each variable were only seen in the wild-type mice (Figure 3) (TPH results for pairwise comparison between trials: Track length: p ≤ 0.001, mdiff = -907.9, serr = 165.88; Velocity: p ≤ 0.001, mdiff = -1.51, serr = 0.28; Activity: p ≤ 0.001, mdiff = -10.03, serr = 1.59). We wanted to know whether this decrease in exploratory activity during the test trial was related to the presence of the stranger mouse or due to reduced drive to explore a now familiar arena. Control experiments were conducted in which the mice explored the arena twice without a stranger mouse present. All other procedures were kept identical to the social tests. These control experiments were conducted only with Ube3am-/p+ (n = 5) and wild-type (n = 5) mice, as we had not found any differences between Ube3am-/p- and Ube3am-/p+ in exploratory activity. During the second trial of the control experiments movement speed, track length and time spent moving decreased for both the Ube3am-/p+ and wild-type mice. The observed decreases were comparable in magnitude to those observed in the social experiments. Decreases in time spent moving reached significance for both Ube3am-/p+ and wild-type mice (F [3,16] = 27.92, p ≤ 0.001; TPH results for pairwise comparison between trials: Ube3am-/p+, p ≤ 0.001, mdiff = -11.1, serr = 2.22, Ube3am+/p+, p ≤ 0.001, mdiff = -11.98, serr = 2.22). Thus, the observed decreases in exploratory activity in the second trial of the social test were not dependent on the presence of a stranger mouse.
Behavior in front of cages
Social interactions with the stranger mouse were quantified as the amount of time the test mice spent in a small area directly in front of the stranger's cage (shaded areas in Figure 1B). Comparison of the total time mice spent directly in front of either cage (duration) revealed that mice of all genotypes spent significantly more time in front of the stranger's cage than in front of the empty cage (Figure 4A) (F [11,272] = 18.62, p ≤ 0.001; TPH results for pairwise comparison between cages during presence of a stranger mouse (test conditions): Ube3am-/p-, p ≤ 0.001, mdiff = 137.7, serr = 28.8; Ube3am-/p+, p ≤ 0.001, mdiff = 136.67, serr = 19.5; Ube3am+/p+, p ≤ 0.001, mdiff = 87.54, serr = 15.92). There were no significant differences between wild type and Ube3a deficient mice for the amount of time spent in front of the stranger's cage. Under control conditions, mice of all genotypes spent the same amount of time in front of both cages, showing that there was no preexisting preference for either cage.
Analysis of the number of visits, i.e. the number of times mice walked into the areas in front of the cages, revealed significant differences between groups (Figure 4B). Under control conditions both Ube3am-/p- and Ube3am-/p+, had significantly fewer visits to either of the two cages than wild-type mice (F [11,272] = 13.96, p ≤ 0.001; TPH results for pairwise comparison with wild-type litter mates for the cage that was empty during control and test trials: Ube3am-/p-, p ≤ 0.015, mdiff = -8.57, serr = 2.33; Ube3am-/p+, p ≤ 0.004, mdiff = -7.15, serr = 1.79. TPH results for pairwise comparison with wild-type litter mates for the cage that was empty during control but housed the stranger mouse during test trials: Ube3am-/p-, p ≤ 0.001, mdiff = -11.94, serr = 2.33; Ube3am-/p+, p ≤ 0.015, mdiff = -8.72, serr = 1.79). Also with the stranger present, Ube3a deficient mice still visited the stranger's cage significantly less often than the wild-type mice (F [11,272] = 13.96; TPH results for pairwise comparison with wild-type litter mates: Ube3am-/p-, p ≤ 0.041 mdiff = -7.84, serr = 2.33, Ube3am-/p+, p ≤ 0.001, mdiff = -8.72, serr = 1.79).
The analysis of the paths within the areas in front of the cages revealed similar differences between Ube3a deficient and wild type mice in movement speed, exploratory path lengths and time spent moving found for the entire path. Ube3a deficient mice spent significantly less time moving in the areas in front of the cages than their wild-type litter mates under control and test conditions (F [11,272] = 20.89, p ≤ 0.001; TPH results for pairwise comparison with wild-type litter mates during control conditions: Ube3am-/p-, p ≤ 0.001, mdiff = -10.72, serr = 2.02; Ube3am-/p+, p ≤ 0.001, mdiff = -9.06, serr = 1.54. TPH results for pairwise comparison with wild-type littermates during test conditions: Ube3am-/p-, p ≤ 0.001, mdiff = -8.73, serr = 2.02; Ube3am-/p+, p ≤ 0.001, mdiff = -7.96, serr = 1.54). There was no significant difference between Ube3am-/p- and Ube3am-/p+ mice in any of the variables. As before, these differences were in not affected by the presence of the stranger mouse.
We also analyzed freezing behavior in the whole field and in front of the cages. There was no significant difference in the number of freezing events between any of the genotypes or between experimental conditions.