Sex-Dependent Motor Deficit and Increased Anxiety-Like States in Mice Lacking Autism-Associated Gene Slit3

Abstract

Altered neuronal connectivity has been implicated in the pathophysiology of Autism Spectrum Disorder (ASD). SLIT/ROBO signaling plays an important role in developmental processes of neuronal connectivity, including axon guidance, neuronal migration, and axonal and dendritic branching. Genetic evidence supports that SLIT3, one of the genes encoding SLITs, is associated with ASD. Yet the causal link between SLIT3 mutation and autism symptoms has not been examined. Here we assessed ASD-associated behaviors in Slit3 knockout (KO) mice. Our data showed that Slit3-KO mice exhibited reduced marble burying behaviors but normal social behaviors. In addition, Slit3-KO mice displayed hypolocomotion in the open field test and impaired motor coordination in the rotarod test. Anxiety-like behaviors were mainly observed in female KO mice assessed by three types of behavioral tests, namely, the open field test, elevated plus maze test, and light/dark box test. No differences were observed between KO and wildtype mice in recognition memory in the novel object recognition test or depression-like behavior in the tail suspension test. Taken together, loss of Slit3 may result in disrupted neural circuits related to motor function and increased anxiety-like states, which are co-occurring symptoms in ASD.

Keywords: Slit3; anxiety; autism(ASD); hypolocomotion; motor coordination.

Figure 1

Slit3-KO mice showed normal social behavior in the three-chamber social test. (A) Both WT and KO mice spent longer time around the cup with a novel mouse than the cup with an object. WT: *p < 0.001, KO: *p < 0.001, t-test. (B) No significant difference in preference index for social stimulus between WT and KO mice. n.s., p = 0.167, t-test. (C) Mice spent longer time around the cup with a novel mouse than with a familiar mouse regardless of their genotypes. WT: *p = 0.012, KO: *p = 0.003, t-test. (D) No significant difference in preference index for social novelty between WT and KO mice. n.s.: p = 0.619, t-test. The preference index was calculated as Equation 1. The data in the bar chart are presented as mean ± SEM. Sample size is indicated in parentheses.

Figure 2

Slit3-KO mice showed suppressed marble burying behavior. (A) Example image of marbles buried by WT and KO mice. (B) KO mice buried significantly fewer marbles compared to WT mice. *p = 0.002, Mann–Whitney test. (C) Number of marbles that female mice buried in each genotype. *p = 0.041, Mann–Whitney test. (D) Number of marbles that male mice buried in each genotype. *p = 0.011, Mann–Whitney test. The data in the bar chart are presented as mean ± SEM. Sample size is indicated in parentheses. F, female; M, male.

Figure 3

Slit3-KO mice were hypolocomotive in the open field test. (A) KO mice traveled a shorter distance than WT mice during a 30-min open field test. *p < 0.001, t-test. (B) When each sex group was analyzed separately, only male KO mice showed hypolocomotion compared to male WT mice. Male: *p < 0.001, Female: n.s.: p = 0.312, t-test. (C) Distance that male WT and KO mice traveled in 5-min time bin. *p = 0.001, genotype effect, two-way rmANOVA. (D) Distance that female WT and KO mice traveled in 5-min time bin. n.s.: p = 0.312, genotype effect, two-way rmANOVA. The data in the bar chart are presented as mean ± SEM. Sample size is indicated in parentheses. F, female; M, male.

Figure 4

Slit3-KO mice displayed impaired motor coordination in the accelerating rotarod test. (A) KO mice showed shorter latency to fall off the rotarod than WT mice. *p = 0.014, genotype effect, two-way rmANOVA. (B) Latency to fall off the rotarod in female mice. *p = 0.023, genotype effect, two-way rmANOVA. (C) Latency to fall off the rotarod in male mice. n.s.: p = 0.477, genotype effect, two-way rmANOVA. *p = 0.008, t-test at the 1st trial. The data are presented as mean ± SEM. Sample size is indicated in parentheses. F, female; M, male.

Figure 5

Slit3-KO mice displayed anxiety-like behaviors during the first 10 mins of the open field test. (A) Duration that each genotype group spent in the center arena of the open field. n.s.: p = 0.614, t-test. (B) Representative maps illustrated mice's track in the open field during the first 10 min of the test. (C) Female KO mice spent less time in the center arena of the open field compared to female WT mice. Female: *p = 0.041, Male: n.s.: p = 0.409, t-test. (D) Distance that animals traveled during the first 10 min of the test. Female: n.s.: p = 0.085, Male: *p = 0.002, t-test. The data in the bar chart are presented as mean ± SEM. Sample size is indicated in parentheses. F, female; M, male.

Figure 6

Slit3-KO mice displayed anxiety-like traits in the elevated plus maze test. (A) KO mice spent less percentage of time in the open arm of the maze compared to WT controls. *p = 0.007, Mann–Whitney test. (B) Heat map illustrating cumulative duration that all mice in each group spent in the elevated plus maze. Open arms are denoted as dash lines, and closed arms are denoted as solid lines. (C) Female WT mice spent more percentage of time in the open arm compared to female KO mice. *p = 0.023, Mann–Whitney test. (D) The percentage of time spent in the open arm was not significantly different between KO and WT male mice. n.s.: p = 0.233, Mann–Whitney test. The data in the bar chart are presented as mean ± SEM. Sample size is indicated in parentheses. F, female; M, male.

Figure 7

Slit3-KO mice showed anxiety-like behaviors in the light/dark box test. (A) KO mice made fewer transitions to the light chamber during the 10-min test than WT controls. *p < 0.001, t-test. (B) Number of transitions to the light compartment in female mice. *p = 0.002, t-test. (C) Number of transitions to the light compartment in male mice. *p = 0.004, t-test. The data in the bar chart are presented as mean ± SEM. Sample size is indicated in parentheses. F, female; M, male.

Figure 8

Slit3-KO mice were not different from WT mice in the novel object recognition test and tail suspension test. (A) There was no significant genotype effect on the novel object discrimination. n.s.: p = 0.340, t-test. Discrimination index was calculated as Equation (2). (B) Immobile duration during the tail suspension test in Slit3-KO mice was not significantly different from WT controls. n.s.: p = 0.972, t-test. The data in the bar chart are presented as mean ± SEM. Sample size is indicated in parentheses.