Cytokine-dependent bidirectional connection between impaired social behavior and susceptibility to seizures associated with maternal immune activation in mice

Abstract

Maternal immune activation (MIA) results in the development of autism in the offspring via hyperactivation of IL-6 signaling. Furthermore, experimental studies showed that the MIA-associated activation of interleukin-1β (IL-1β) concurrently with IL-6 increases the rate and the severity of hippocampal kindling in mice, thus, offering an explanation for autism-epilepsy comorbidity. We examined whether epileptic phenotype triggered by prenatal exposure to IL-6 and IL-1β combination is restricted to kindling or whether it is reproducible in another model of epilepsy, whereby spontaneous seizures develop following kainic acid (KA)-induced status epilepticus. We also examined whether in mice prenatally exposed to IL-6 and IL-6+IL-1β, the presence of spontaneous seizures would exacerbate autism-like features. Between days 12 and 16 of pregnancy, C57BL/6J mice received daily injections of IL-6, IL-1β, or IL-6+IL-1β combination. At postnatal day 40, male offspring were examined for the presence of social behavioral deficit, and status epilepticus was induced by intrahippocampal KA injection. After 6weeks of monitoring for spontaneous seizures, sociability was tested again. Both IL-6 and IL-6+IL-1β offspring presented with social behavioral deficit. Prenatal exposure to IL-6 alleviated, while such exposure to IL-6+IL-1β exacerbated, the severity of KA-induced epilepsy. Increased severity of epilepsy in the IL-6+IL-1β mice correlated with the improvement of autism-like behavior. We conclude that complex and not necessarily agonistic relationships exist between epileptic and autism-like phenotypes in an animal model of MIA coupled with KA-induced epilepsy and that the nature of these relationships depends on components of MIA involved.

Keywords: Autism; Comorbidity; Cytokines; Epilepsy; Interleukin-1β; Interleukin-6; Maternal immune activation.

Fig. 1. An example of spontaneous seizure after kainic acid (KA) - induced status epilepticus

Spontaneous seizure was captured in a 12 week-old mouse five weeks after intrahippocampal KA injection (50 ng). Top two rows. Snapshots of a stage 4 spontaneous seizure. Time is indicated from the seizure onset. Note the progression from tonic phase (3 s) to clonic-tonic phase (“kangaroo” posture, 9-15 s). Also, note postictal depression lasting for over 40 s (22 s and 66 s samples), evident as retaining same position without movements. Bottom row. Electrographic seizure from the same mouse. Note postictal depression.

Fig. 2. Performance in the three-chamber sociability test

Left. The first test was performed between P35 and P40 prior to KA administration. The offspring of saline-treated and IL-1β- treated mice showed strong preference towards the conspecific vs. the unanimated object. In the IL-6 and IL-6+IL-1β offspring, sociability was significantly diminished to the levels of indifference. Right. The second test was performed ten weeks after KA SE induction, upon the establishment of spontaneous seizures at the age of 16 weeks. In Saline/KA and IL-1β/KA animals the sociability remained intact (i.e. was similar to the one prior to the KA injection). In IL-6/KA mice, social behavior was diminished, but the impairment was not further exacerbated by recurrent seizures. In IL-6+IL-1β/KA animals, sociability was significantly improved as compared with pre-epilepsy level, and was statistically similar to the one in normal (i.e. Saline/Saline) mice. Data are shown as Mean±SEM. *-p<0.05 vs respective Saline; †- p p<0.05 “After KA” vs. “Before KA”. 2-way ANOVA followed by Sidak's multiple comparisons test. Interaction F=11.52 (p<0.05); effect of seizures F=4.46, p<0.05; effects of prenatal treatment F=28.26, p<0.05.

Fig. 3. Spontaneous seizures

Average daily count of spontaneous seizures was derived from 6 weeks-long seizure monitoring. IL-6/KA mice developed significantly fewer, and IL-6+IL-1β/KA mice- significantly more spontaneous seizures than Saline/KA animals. Occasional rare spontaneous seizures were observed after intrahippocampal saline injection in animals of all groups. Data are shown as Mean±SEM. *-p<0.05 vs Saline+KA. Kruskal-Wallis followed by Mann-Whitney test.

Fig. 4. NeuN- positive cell counts in the hippocampus

A. NeuN positive cell counts after prenatal cytokine exposure only. Administration of neither of the cytokines, nor of their combination produced neurodegeneration in the hippocampus. Ipsilateral and Contralateral refer to the site of saline injection. Data are shown as Mean±SEM per cent of NeuN-positive cell counts in the offspring of saline-treated mice. 2-Way ANOVA+Sidak's multiple comparison test. Interaction F=1.346, p>0.05; effects of prenatal treatment F=0.703, p>0.05. B. Examples of NeuN immunostaining a Saline/Saline (noted as Saline) and three different Saline/KA (noted as KAx) mice. In Saline and KA2 animals, left (injected) hippocampi, and in KA1 and KA3- hippocampi contralateral to the injection are shown. Various degrees of neurodegeneration in the CA1, CA3 and hilar areas are observed. KA1- very mild CA1 and CA3 cell loss; KA2- moderate CA3 injury; KA3- moderate CA1 and hilus injury. Scale bar- 500 uM. C. NeuN-positive cell counts after intrahippocampal KA administration. Administration of neither of the cytokines, nor of their combination exacerbated KA-induced neuronal cell loss in the hippocampus. Ipsilateral and Contralateral refer to the site of KA injection. Data are shown as Mean±SEM per cent of NeuN-positive cell counts in the offspring of Saline/KA mice. 2-Way ANOVA+Sidak's multiple comparison test. Interaction F=0.69, p>0.05; effects of prenatal treatment F=1.02, p>0.05; effects of seizures F=0.41, p>0.05.