Sex-Selective Effects on Behavior in a Mouse Model of Tuberous Sclerosis Complex

Abstract

Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder that is caused by a mutation in either TSC1 or TSC2 TSC affects multiple systems of the body, and patients with TSC display a range of neurologic and behavioral manifestations including seizures, intellectual disability, autism spectrum disorders, attention deficit hyperactivity disorder, anxiety, and mood disorders. Whereas behavioral phenotypes of many mouse models have been studied, the effects of sex have, for the most part, not been explored. We studied adult male and female Tsc2 heterozygous and control mice to investigate the influence of sex and genotype on behavior. On a test of social preference, Tsc2 heterozygous mice, regardless of sex, demonstrated lower preference for the stranger mouse than control mice. In the open field, Tsc2 heterozygous males and control females habituated to the open field with decreasing anxiety-like behavior over time, whereas Tsc2 heterozygous females did not show habituation to the open field environment. We did not find any statistically significant effects of genotype on open field activity, learning and memory or motor function. Our results highlight phenotype differences in Tsc2 heterozygous mice, some of which are influenced by sex. A consideration of how sex influences the behavioral phenotypes of TSC is critical to develop a more complete understanding of the disorder and better target future pharmacological treatments.

Keywords: sex differences; tuberous sclerosis complex.

Figure 1.

Timeline of testing. We started with two cohorts of mice.

Figure 2.

Activity levels. In distance traveled on the open field, we did not find any statistically significant main effects or interactions. Each point represents the mean ± SEM for the number of animals indicated in parentheses.

Figure 3.

Anxiety-like behavior. A, In the open field, we analyzed the ratio of distance traveled in the center of the open field to total distance traveled. The genotype × sex × epoch interaction was statistically significant. In control mice, males traveled more relative distance in the center than females in epochs 1 and 2 (p = 0.002 for each epoch). In Tsc2 heterozygous mice, males traveled more relative distance in the center than females in epochs 2, 3, and 5 (p = 0.049, p = 0.015, and p = 0.008, respectively). Each point represents the mean ± SEM for the number of mice indicated in parentheses. B, We also analyzed anxiety-like behavior with the zero maze by measuring the time in the open portion of the maze. There were no statistically significant interactions or main effects. Bars represent mean ± SEM for the number of animals indicated in parentheses.

Figure 4.

Social preference. A, The chamber × genotype interaction approached statistical significance (p = 0.052). All mice, regardless of sex or genotype, sniffed the stranger mouse more than the object. Post hoc tests showed that control mice, regardless of sex, sniffed the social mouse more than Tsc2 heterozygous mice did (p = 0.016). Bars represent mean ± SEM for the number of mice indicated in parentheses. B, In the preference for social novelty test, the genotype × sex × chamber interaction was statistically significant (p = 0.011). Only Tsc2 heterozygous females sniffed the novel mouse statistically significantly more than the familiar mouse (p < 0.001; paired post hoc tests). In males, Tsc2 heterozygous mice spent less time sniffing the novel mouse compared with control mice (p = 0.002). In females, Tsc2 heterozygous mice spent more time sniffing the novel mouse compared with control mice (p = 0.018). Other statistically significant effects (not indicated on the figure) are that control male mice spent more time sniffing both the familiar (p = 0.013) and novel (p = 0.006) mice compared with control female mice, and Tsc2 heterozygous male mice spent less time sniffing the novel mouse than Tsc2 heterozygous females (p = 0.005). Bars represent mean ± SEM for the number of mice indicated in parentheses. *p < 0.05.

Figure 5.

Learning and memory. A, Results of NOR test do not show any statistically significant interaction or main effects. Bars represent mean ± SEM of the number of mice indicated in parentheses. B, In the passive avoidance test, the main effect of sex was statistically significant (p = 0.036), but effects of genotype were not. Overall, regardless of genotype, females had a lower latency to enter the dark side than males. Bars represent mean ± SEM for the number of animals indicated in parentheses. The legend in B applies to both panels.

Figure 6.

Motor function. A, Average latencies to fall off an accelerating RotaRod after two trials do not show any statistically significant interactions or main effects. Bars represent mean ± SEM for the number of animals indicated in parentheses. B, C, Analysis of gait (step length and step width) show a statistically significant main effect of measure (p < 0.001) indicating that step width is less than step length. There were no statistically significant differences regarding sex or genotype. Bars represent mean ± SEM for the number of animals indicated in parentheses. The legend in A applies to all three panels.

Figure 7.

Social preference in control males in the presence or absence of the experimenter. We compared sociability and preference for social novelty whether or not the experimenter was present in the room (note: the male experimenter differed for each condition). A, The experimenter presence × chamber interaction was not statistically significant, but both the main effect of experimenter presence (F_(1,45) = 243.1, p < 0.0001) and chamber (F_(1,45) = 18.72, p < 0.0001) were. B, The experimenter presence × chamber interaction was statistically significant (F_(1,44) = 8.35, p = 0.006). Post hoc t tests showed that control males have a statistically significant preference for the novel mouse compared with the familiar mouse (p < 0.001) only if the experimenter is present. Bars represent mean ± SEM for the number of mice indicated in parentheses. We tested 26 mice with the experimenter present and 22 mice with the experimenter absent. Data from one mouse during the sociability phase was lost. During the social novelty phase with the experimenter present, we lost one mouse because the door closed on his tail following habituation and another mouse because someone entered the room during the test. *p < 0.001.