Early-life febrile seizures worsen adult phenotypes in Scn1a mutants

Abstract

Mutations in the voltage-gated sodium channel (VGSC) gene SCN1A, encoding the Na_v1.1 channel, are responsible for a number of epilepsy disorders including genetic epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome (DS). Patients with SCN1A mutations often experience prolonged early-life febrile seizures (FSs), raising the possibility that these events may influence epileptogenesis and lead to more severe adult phenotypes. To test this hypothesis, we subjected 21-23-day-old mice expressing the human SCN1A GEFS+ mutation R1648H to prolonged hyperthermia, and then examined seizure and behavioral phenotypes during adulthood. We found that early-life FSs resulted in lower latencies to induced seizures, increased severity of spontaneous seizures, hyperactivity, and impairments in social behavior and recognition memory during adulthood. Biophysical analysis of brain slice preparations revealed an increase in epileptiform activity in CA3 pyramidal neurons along with increased action potential firing, providing a mechanistic basis for the observed worsening of adult phenotypes. These findings demonstrate the long-term negative impact of early-life FSs on disease outcomes. This has important implications for the clinical management of this patient population and highlights the need for therapeutic interventions that could ameliorate disease progression.

Keywords: Epilepsy; Febrile seizures; GEFS+; Na(v)1.1; SCN1A.

Figure 1. RH mutants show greater susceptibility to hyperthermia-induced seizures

A. Susceptibility to acute hyperthermia-induced seizures was evaluated at 3 different ages: P14-15, P22-24, and P32-33. B. Seizure severity, based on a modified Racine scale, was determined.

Figure 2. EEG activity during acute FS induction in P14 mice

Representative EEG recordings during hyperthermia: (A) P14 WT mouse, (B) P14 RH/+ mutant with behavioral seizure, and (C) P14 RH/+ mutant without behavioral seizure. Arrows indicate seizure activity. Two cortical electrodes EEG₁ and EEG₂ were each referenced the fourth cortical electrode (EEG₄) to generate the EEG – REF montage: (e.g. EEG₁-EEG₄).

Figure 3. Acute FS generation is associated with increased neuronal activity in the hippocampus

Representative c-Fos immunoreactivity in the dentate gyrus (DG), thalamus (paraventricular thalamic nucleus (PVT)), and locus coeruleus (LC) from P22 WT and RH/+ mice 2 hours post-acute seizure induction. 40X magnification, scale bar, 100 µm.

Figure 4. Early-life FSs increase susceptibility to flurothyl-induced seizures in adult RH/+ mutants

The latencies to the (A) myoclonic jerk (MJ), (B) the first generalized tonic clonic seizure (GTCS), and (C) the generalized tonic clonic seizure with hindlimb extension (GTCS-HE) were compared between WT and RH/+ mice subjected to the PFE and A/PFE paradigms and control mice. * p < 0.05, ** p < 0.001. Data are shown as mean ± standard error of the mean (SEM).

Figure 5. A/PFE paradigm results in behavioral alterations in adult RH/+ mice

(A-B) Open Field test: (A) RH/+ mice exposed to the A/PFE move at faster speeds and (B) travel greater distances in an open field compared to WT mice subjected to the A/PFE and control WT and RH/+ mice. (C-D) 3-Chamber Social Interaction test: (C) A/PFE RH/+ mice spent more time with the stranger mouse when compared to the empty container, but the difference was not statistically significant. (D) A/PFE RH/+ mice spent comparable amounts of time with the familiar and novel mouse. (E) Object Recognition Test: RH/+ mice subjected to the A/PFE did not show a preference for the novel object, resulting in a discrimination index of 47 ± 5.4%. * p < 0.05. Data are shown as mean ± standard error of the mean (SEM).

Figure 6. A/PFE paradigm causes an increase in firing rates of mutant pyramidal CA3 neurons

(A) The firing patterns of pyramidal neurons from each of the 4 groups of mice are shown at current injections of 10 pA, 90 pA and 170pA. (B) The number of APs fired in a 2-second period is plotted against the corresponding current injection. Data are shown as mean ± SEM; n = number of cells. Statistical significance was determined by 2-way ANOVA, Holm-Sidak correction. RH/+ mutants subjected to the A/PFE were significantly different (p < 0.05) from all other groups.