Deletion of Fmr1 results in sex-specific changes in behavior

Abstract

Objective: In this study, we used a systemic Fmr1 knockout in order to investigate both genotype- and sex-specific differences across multiple measures of sociability, repetitive behaviors, activity levels, anxiety, and fear-related learning and memory.

Background: Fragile X syndrome is the most common monogenic cause of intellectual disability and autism. Few studies to date have examined sex differences in a mouse model of Fragile X syndrome, though clinical data support the idea of differences in both overall prevalence and phenotype between the sexes.

Methods: Using wild-type and systemic homozygous Fmr1 knockout mice, we assessed a variety of behavioral paradigms in adult animals, including the open field test, elevated plus maze, nose-poke assay, accelerating rotarod, social partition task, three-chambered social task, and two different fear conditioning paradigms. Tests were ordered such that the most invasive tests were performed last in the sequence, and testing paradigms for similar behaviors were performed in separate cohorts to minimize testing effects.

Results: Our results indicate several sex-specific changes in Fmr1 knockout mice, including male-specific increases in activity levels, and female-specific increases in repetitive behaviors on both the nose-poke assay and motor coordination on the accelerating rotarod task. The results also indicated that Fmr1 deletion results in deficits in fear learning and memory across both sexes, and no changes in social behavior across two tasks.

Conclusion: These findings highlight the importance of including female subjects in preclinical studies, as simply studying the impact of genetic mutations in males does not yield a complete picture of the phenotype. Further research should explore these marked phenotypic differences among the sexes. Moreover, given that treatment strategies are typically equivalent between the sexes, the results highlight a potential need for sex-specific therapeutics.

Keywords: Fragile X syndrome; anxiety; autism; phenotypes; plasticity.

Figure 1

Deletion of Fmr1 resulted in sex‐specific hyperactivity in the open field task. (a) Fmr1 KOs showed significant hyperactivity when examining distance moved in the open field task. Upon further statistical analysis, hyperactivity was only detected in the male knockouts. (b) When exploratory behavior was analyzed in 10‐min epochs, Fmr1 knockouts show similar degrees of habituation during the testing window, however, are more active overall. (c) Fmr1 KOs show increased activity levels specifically in the periphery of the testing apparatus. (d) Male Fmr1 KOs exhibited significantly higher amounts of rearing behavior compared to wild‐types, whereas this effect was not present in females. Data are presented as mean ± SEM. ** = p < .01, *** = p < .001

Figure 2

Females displayed decreased anxiety in the elevated plus maze task, independent of genotype. Females across genotypes display less anxiety than their male counterparts, as females spent more time in the open arm as a group compared to males. Data are presented as mean ± SEM. * = p < .05

Figure 3

Deletion of Fmr1 resulted in sex‐specific increases in repetitive behavior across two tasks. (a), Females, independent of genotype, showed a shortened latency to initiate a nose poke than their male counterparts. (b) Analysis of the hole‐poking behavior as a function of center versus surround demonstrated that female Fmr1 KOs displayed an increase in hole‐poking behavior in the outer holes, whereas male Fmr1 KOs did not display this increase (c) Female Fmr1 KOs exhibited higher amounts on hole‐poking behavior on corner holes poked, whereas male Fmr1 KOs did not differ significantly from male WTs. Data are presented as mean ± SEM.*  = p < .05; ** = p < .01

Figure 4

Fmr1 knockout females display enhanced motor coordination on the accelerating rotarod task. Female KO mice showed enhanced latency to fall on later trials of the accelerating rotarod task. Data are presented as mean ± SEM. * = p < .05

Figure 5

Fmr1 knockouts displayed decreased learning in the trace fear conditioning task. (a) Fmr1 KOs exhibited less freezing behavior during the acquisition of the fear response across the six trace periods. (b) Fmr1 KOs exhibited less freezing during the trace period in a novel testing environment. Data are presented as mean ± SEM. * = p < .05; ** = p < .01

Figure 6

Fmr1 knockouts displayed decreased learning and memory capabilities in the delayed fear conditioning task. (a) Fmr1 KOs presented significantly decreased freezing in response to the first intertrial interval (ITI), second CS presentation and second ITI. (b) No significant differences by sex and genotype were detected in contextual fear conditioning. (c) Fmr1 KOs exhibited significantly reduced freezing to presentation of the CS in a novel context. Data are presented as mean ± SEM. * = p < .05