Increased susceptibility to kainate-induced seizures in a mouse model of tuberous sclerosis complex: Importance of sex and circadian cycle

Abstract

Objective: Comorbidity of epilepsy and autism in tuberous sclerosis complex 2 (TSC2) is very frequent, but the link between these conditions is still poorly understood. To study neurological problems related to autism, the scientific community has been using an animal model of TSC2, Tsc2^+/- mice. However, it is still unknown whether this model has the propensity to exhibit increased seizure susceptibility. Further, the importance of sex and/or the circadian cycle in this biological process has never been addressed. This research aimed to determine whether male and female Tsc2^+/- mice have altered seizure susceptibility at light and dark phases.

Methods: We assessed seizure susceptibility and progression in a Tsc2^+/- mouse model using the chemical convulsant kainic acid (KA), a potent agonist of the AMPA/kainate class of glutamate receptors. Both male and female animals at adult age were evaluated during non-active and active periods. Seizure severity was determined by integrating individual scores per mouse according to a modified Racine scale. Locomotor behavior was monitored during control and after KA administration.

Results: We found increased seizure susceptibility in Tsc2^+/- mice with a significant influence of sex and circadian cycle on seizure onset, progression, and behavioral outcomes. While, compared to controls, Tsc2^+/- males overall exhibited higher susceptibility independently of circadian cycle, Tsc2^+/- females were more susceptible during the dark and post-ovulatory phase. Interestingly, sexual dimorphisms related to KA susceptibility were always reported during light phase independently of the genetic background, with females being the most vulnerable.

Significance: The enhanced susceptibility in the Tsc2 mouse model suggests that other neurological alterations, beside brain lesions, may be involved in seizure occurrence for TSC. Importantly, our work highlighted the importance of considering biological sex and circadian cycle for further studies of TSC-related epilepsy research.

Plain language summary: Tuberous sclerosis complex (TSC) is a rare genetic disorder. It causes brain lesions and is linked to epilepsy, intellectual disability, and autism. We wanted to investigate epilepsy in this model. We found that these mice have more induced seizures than control animals. Our results show that these mice can be used in future epilepsy research for this disorder. We also found that sex and time of day can influence the results. This must be considered in this type of research.

Keywords: autism spectrum disorder; biological sex; circadian cycle; epilepsy; tuberous sclerosis complex.

FIGURE 1

Schematic representation of experimental protocol used to characterize seizure susceptibility and severity to kainic acid. Male and female mice aged between 8 and 12 weeks were individually placed in an open‐field box (20 × 40 cm), video‐recorded (from top and front), and evaluated over 120 min: 20 min at control conditions following saline intraperitoneal (i.p.) injection and 100 min after a single kainic acid (KA) injection (20 mg/kg, i.p.). The figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license.

FIGURE 2

Acute KA administration significantly increases vulnerability in the TSC2 mouse model. The Racine scores were evaluated during 120 min of the experiment, following acute injection of saline and KA solution at (A) light and (B) dark phases. The red dashed line represents the time of KA injection, the light orange rectangular frame the time chosen to study seizure severity during stabilization of seizure occurrence (80–100 min), and the symbols are the mean values every 10 min between 20′ and 120′ (10 time points). For saline conditions, all mice exhibited normal behavior (Racine scores = 0) that changed post‐KA to a pattern of seizure events highlighted by susceptibility of Tsc2 ^+/− animals for both light and dark phase. Post hoc tests following ANOVA revealed a sex‐dependent vulnerability for both light and dark periods as only Tsc2 ^+/− males had significantly higher scores than WT males. Moreover, during the light phase in control animals, females exhibited significantly increased severity by a constant pattern of higher Racine scores compared with WT males following KA injection. The results are expressed as mean ± SEM (n = 12 for each group). *p < 0.05, **p < 0.01 comparing WT and Tsc2 ^+/− males and ^& p < 0.05, ^&& p < 0.01 comparing WT males and WT females by 2WAY repeated measures ANOVA with Tukey's multiple comparisons‐test.

FIGURE 3

KA administration increases seizure vulnerability for Tsc2 ^+/− mice in a sex and circadian cycle‐dependent matter. (A) Latency to the first seizure accordingly to each circadian cycle. Overall mutant mice had shorter latency times than their WT littermates that occurred in males for both circadian phases and in females during the dark phase. In control animals, males exhibited significantly higher latency times than females for the light phase. (B) Racine score values accordingly to each circadian cycle. Males during light phase showed a statistically significant difference between experimental groups, exposing an increased susceptibility for Tsc2 ^+/− males compared with their WT littermates. (C) Number of seizures accordingly to each circadian cycle. Tsc2 ^+/− females had higher number of seizures compared with their WT littermates during dark phase and compared with Tsc2 ^+/− male during light phase. Moreover, for Tsc2 ^+/− males, number of seizures was significantly increased during dark phase compared with light phase. The results are expressed as mean ± SEM (n = 8–12 for each group). *p < 0.05, **p < 0.01, ***p < 0.001 by 3‐WAY ANOVA with Sidak's multiple comparisons test correction.

FIGURE 4

Exploratory behavior decreases significantly and similarly post‐KA injection across all groups. (A) Total distance traveled for each circadian cycle and condition (saline and KA). All animals at saline conditions had similar distances traveled in open field arena that decreased significantly post‐KA injection at both light and dark phase. (B) Total distance traveled for each circadian cycle for KA condition. No significant differences were reported between experimental groups post‐KA injection. The results are expressed as mean ± SEM (n = 10–12 for each group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Ordinary 1‐WAY ANOVA (A) and 3‐WAY ANOVA (B) with Sidak's multiple comparisons test.

FIGURE 5

Anxiety levels post‐KA are influenced by sex and circadian cycle in a genotype‐dependent manner. (A) Percentage of time in the center of the open field for each circadian cycle and condition (saline and KA). Tsc2 ^+/− animals showed a significant increase of anxiety‐like behavior, comparing saline and post‐KA condition, that generally was not observed for WT mice. (B) Percentage of time in the center of the open field for each circadian cycle for KA condition. WT females exhibited lower anxiety levels during dark phase compared with Tsc2 ^+/− females. The results are expressed as mean ± SEM (n = 8–12 for each group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Ordinary 1‐WAY ANOVA (A) and 3‐WAY ANOVA (B) with Sidak's multiple comparisons test.

FIGURE 6

Estrous cycle influenced females' severity to KA in a genotype‐dependent manner. (A) Latency to the first seizure accordingly to each circadian cycle. Latency to the first seizure was significantly reduced for Tsc2 ^+/− females compared with WT animals at the both ovulatory and post‐ovulatory phases during the dark phase. (B) Racine score values accordingly to each circadian cycle. Racine scores were significantly increased in the mutant female compared with WT at the post‐ovulatory stage during the dark phase. Moreover, in control animals, Racine scores were significantly higher in the ovulatory phase compared with post‐ovulatory phase during dark phase and during light phase compared with dark phase for post‐ovulatory phase. (C) Number of seizures accordingly to each circadian cycle. Tsc2 ^+/− females in post‐ovulatory stage had higher number of seizures compared with their WT group during dark phase. Moreover, for WT animals in post‐ovulatory phase, number of seizures was significantly increased during light phase compared with dark phase. The results are expressed as mean ± SEM (n = 4–8 for each group). *p < 0.05, ***p < 0.001, ****p < 0.0001 by 3‐WAY ANOVA with Sidak's multiple comparisons test.