We used two lines of mice with non-identical genetic backgrounds (see Materials and Methods, Mice). One line had a mixed genetic background, referred to as M mice. A second mouse line was created by backcrossing M HT mice to 129X1/SvJ mice, here referred to as 129 enriched (129E) mice.
The Sept5 genotype (i.e. KO, HT and their two WT pair groups) affected active social interaction (F3,94 = 13.96, P < 0.01) and the genotype effect had a significant interaction with genetic backgrounds (F3,94 = 3.05, P < 0.05) (Fig. A). Newman–Keuls comparisons confirmed that M KO mice exhibited lower levels of active social interaction than M WT mice, but this effect failed to reach significance in 129E mice. 129E mice generally exhibited higher levels of active social interaction than M mice (F1,94 = 199.94, P < 0.01). Under our experimental condition, mice showed low, almost negligible levels of passive social interaction (Fig. B). Although M mice had higher levels of passive social interaction than 129E mice (F1,94 = 4.18, P < 0.05), the four genotype groups did not differ (F3,94 = 1.61, n.s.), even when genetic background was taken into account (F3,94 = 0.47, n.s.).
Figure 1. Active (A) and passive (B) social interactions. Interaction time (mean ± SEM) is shown in two successive 5 min sessions. Asterisks indicate statistically significant differences from WT mice at 1% (**) levels, as determined by Newman–Keuls (more ...)
To further explore the nature of defective active social interaction in M KO mice, we divided active social interaction into aggressive behaviors and non-aggressive, affiliative social behaviors. Because our experimental condition was designed to optimally evaluate non-aggressive, affiliative social interaction, it was much more common than sporadic and negligible aggressive behaviors (F1,30 =37.55, P < 0.01; data not shown). M WT mice showed significantly more affiliative active social interaction than M KO mice (F1,30 = 10.22, P < 0.01, data not shown), but the two groups did not differ in the amount of aggressive behaviors (F1,30 = 2.74, n.s., data not shown).
Olfactory investigation was the predominant affiliative active social interaction in M WT and M KO mice. However, a deficit in affiliative active social interaction seen in M KO mice does not seem to reflect a generalized deficit in olfactory sensing, as M WT and M KO mice did not differ in avoidance of a compartment containing a non-social olfactory stimulus (2-methylbutyric acid, 1.7 × 10−4 mol) [t(10) = 0.23, n.s., data not shown].
Elevated plus maze
In the elevated plus maze, Sept5 deficient mice spent more time in normally avoided open arms than WT littermates (F2,121 = 3.32, P < 0.05), regardless of genetic background (Genotype×Genetic background interaction, F2,121 = 0.13, n.s.) (Fig. A). The significant genotype effect was mainly due to higher levels of time spent in the open arms in HT mice. The 129E mice spent more time in the open arms than M mice (F1,121 = 7.63, P < 0.01). The number of entries to open arms was indistinguishable among genotypes (F2,121 = 2.19, n.s.).
Figure 2. Anxiety- and affect-related traits. (A) Elevated plus maze. The relative amount of time (% mean ± SEM) spent in the open arms is shown. Because the homogeneity of variance was violated (Hartley’s F-max = 57.87, P < 0.01), data (more ...)
Mice placed in an inescapable open field tend to stay in the vicinity of the walls. This tendency or thigmotaxis is interpreted as reflecting, in part, an anxiety- or stress-related trait. Thigmotaxis was not affected by genotype (F2,118 = 1.01, n.s.) or genetic background (F1,118 = 0.79, n.s.) (Fig. B). Genotype had no interaction with genetic background (F2,118 = 2.03, n.s.), time (F10,590 = 1.57, n.s.) or genetic background and time (F10,590 = 1.42, n.s.).
When rodents are held upside-down by the tail, they exhibit mobility in an apparent attempt to escape from the situation. This behavior, or its absence (i.e. immobility), is thought to reflect a basal affective state and a stress response (41
). Immobility was not affected by genotype (F2,158
= 1.04, n.s.), genetic background (F1,158
=v3.39, n.s.) or interaction between genetic background and genotype (F2,158
= 2.23, n.s.) (Fig. C).
A startle response was examined against pulses ranging from 75 to 120 dB. Mice exhibited a startle response at high stimulus levels (F9,1215 = 156.12, P < 0.01) (Fig. A). Although the intensity of startle was not affected by genotype (F2,135 = 0.02, n.s.) or by interaction between genotype and genetic background (F2,135 = 1.72, n.s.), M mice showed basally higher startle responses than 129E mice at high pulse levels (genetic background, F1,135 = 30.77, P < 0.01; pulse×genetic background, F9,1215 = 29.28, P < 0.01).
Figure 3. Startle (A) and PPI (B). The magnitude of a startle response and the percentage of PPI are plotted against pulse stimuli (75–120 dB) and prepulse stimuli at 4, 8, 12 and 16 dB (69, 73, 77 and 81 dB presented with the 65 dB background noise), respectively. (more ...)
In rodents and humans, a startle response evoked by a loud sound is reduced by the presentation of a preceding non-startle stimulus, a process termed PPI. PPI increased as the prepulse intensity was increased (F3,405 = 51.53, P < 0.01) (Fig. B). KO mice showed higher levels of PPI than WT or HT mice (F2,135 = 4.27, P < 0.05). Higher levels of basal PPI were found in M mice than in 129E mice (F1,135 = 8.78, P < 0.01). M mice showed a higher rate of PPI increase across prepulse levels than did 129E mice (F3,405 = 2.64, P < 0.05).
When mice were trained to run on an L-maze to reach a food-baited goal compartment, they gradually learned to reach the goal faster (Fig. A) (F5,4310 = 51.03, P < 0.01). Because the three-way interaction was significant (F10,4310 = 2.50, P < 0.01), we applied exploratory two-way analysis of variances (ANOVAs) to the two genetic backgrounds separately. In M mice, Sept5 deficiency delayed, in a gene-dose dependent manner, acquisition of this behavior evenly across sessions (Genotype, F2,405 = 6.26, P < 0.01; genotype×session, F10,2025 = 1.73, n.s.). In contrast, Sept5 deficiency in 129E mice delayed and facilitated this behavior in KO mice and HT mice, respectively, at early sessions only (genotype, F2,457 = 1.22, n.s.; genotype×session, F10,2285 = 3.64, P < 0.01).
Figure 4. (A) Rewarded approach. Time (mean ± SEM) spent to reach a rewarded goal in the L-maze is shown. M group: WT, n = 19; HT, n = 32; KO, n = 17. 129E group: WT, n = 23; HT, n = 36; KO, n = 18. (B) Spontaneous alternation. All genotypes show a tendency (more ...)
In a T-maze, mice have a tendency to visit an unvisited arm rather than a previously visited arm. This phenomenon or spontaneous alternation reflects the flexibility of an animal to alternate their behavior based on the memory of a previously visited arm. Spontaneous alternation was not influenced by genetic background (F1,141 = 0.01, n.s.), genotype (F2,141 = 1.04, n.s.) or interaction (F2,141 = 2.25, n.s.) (Fig. B).
When mice are rewarded in one of the two goal arms of the T-maze, their tendency to subsequently alternate behavior is enhanced. This reward-based memory or rewarded alternation is an objective measure to evaluate how a reward experience subsequently influences spontaneous alternation (Fig. C). Sept5 deficiency had no effect on rewarded alternation by itself (F2,93 = 0.07, n.s.), with genetic background (F2,93 = 2.09, n.s.) or with genetic background and session (F8,372 = 1.22, n.s.). 129E mice exhibited higher levels of rewarded alternation than M mice (F1,93 = 8.47, P < 0.01).
Spontaneous motor activity
Many of the affective and cognitive behaviors could be confounded by altered basal levels of motor activity. We examined spontaneous locomotor activity in an inescapable open field. Mice exhibited a normal rate of habituation regardless of genetic background (time interval, F5,590 = 113.87, P < 0.01; genetic background×time interval, F5,590 = 1.78, n.s.) (Fig. ). Sept5 deficiency had no effect on motor activity at any time point, regardless of genetic background (genotype, F2,118 = 1.83, n.s; genotype×genetic background, F2,118 = 1.33, n.s.; genotype×time interval, F10,590 = 0.36, n.s.; genotype×genetic background×time interval, F10,590 = 1.11, n.s.). Overall, M mice exhibited higher levels of basal motor activity than 129E mice (genetic background, F1,118 = 20.53, P < 0.01).
Figure 5. Spontaneous locomotor activity in an inescapable open field. Distance traveled (mean ± SEM) was measured for 30 min. Each tick on the x-axis represents a 5 min bin. WT, wild-type mice; KO, Sept5 knockout mice. M group: WT, n = 18; HT, n = 19; (more ...)