Role of the hippocampus in Nav1.6 (Scn8a) mediated seizure resistance

Abstract

SCN1A mutations are the main cause of the epilepsy disorders Dravet syndrome (DS) and genetic epilepsy with febrile seizures plus (GEFS+). Mutations that reduce the activity of the mouse Scn8a gene, in contrast, are found to confer seizure resistance and extend the lifespan of mouse models of DS and GEFS+. To investigate the mechanism by which reduced Scn8a expression confers seizure resistance, we induced interictal-like burst discharges in hippocampal slices of heterozygous Scn8a null mice (Scn8a(med/+)) with elevated extracellular potassium. Scn8a(med/+) mutants exhibited reduced epileptiform burst discharge activity after P20, indicating an age-dependent increased threshold for induction of epileptiform discharges. Scn8a deficiency also reduced the occurrence of burst discharges in a GEFS+ mouse model (Scn1a(R1648H/+)). There was no detectable change in the expression levels of Scn1a (Nav1.1) or Scn2a (Nav1.2) in the hippocampus of adult Scn8a(med/+) mutants. To determine whether the increased seizure resistance associated with reduced Scn8a expression was due to alterations that occurred during development, we examined the effect of deleting Scn8a in adult mice. Global Cre-mediated deletion of a heterozygous floxed Scn8a allele in adult mice was found to increase thresholds to chemically and electrically induced seizures. Finally, knockdown of Scn8a gene expression in the adult hippocampus via lentiviral Cre injection resulted in a reduction in the number of EEG-confirmed seizures following the administration of picrotoxin. Our results identify the hippocampus as an important structure in the mediation of Scn8a-dependent seizure protection and suggest that selective targeting of Scn8a activity might be efficacious in patients with epilepsy.

Keywords: Cre recombinase; Dravet syndrome; GEFS+; Lentivirus; Na(v)1.6; Nav1.1; Scn1a; Scn8a; Voltage-gated sodium channel.

Figure 1

Spontaneous seizure-like burst discharges in CA3 pyramidal cell layer induced by elevated potassium in hippocampal slices from Scn8a^med/+ mutant and WT mice. Aa, CA3 pyramidal cells did not show spontaneous burst activity in physiological 2.5 mM [K⁺]_o; however, burst activity was induced when extracellular K⁺ concentration was elevated to 8.5 mM [K⁺]_o. Ab, Expansion of a segment of spontaneous activity showed synchronous firing of burst activity. Ac, Expansion of a single interictal-like burst discharge. Arrow indicates start of a burst discharge and star shows the end of a single burst. B, Extracellular recording from CA3 pyramidal cell layer of a Scn8a^med/+ mutant mouse (P24). C, Population spike amplitude before and after exposing hippocampal slice to 8.5 mM [K⁺]_o. Solid gray – before perfusing 8.5 mM [K⁺]_o into the bath. Black dash – after exposing slice to 8.5 mM [K⁺]_o and washout with standard 2.5 mM [K⁺]_o ACSF. Population spikes were evoked with 50 αA stimulation for 100 μs. D, Hippocampal slices from Scn8a^med/+ mutant and WT mice from P13-P20 demonstrated comparable levels of spontaneous burst discharge activity in elevated extracellular potassium. Beginning at P21, the propensity for burst activity dramatically decreased in hippocampal slices from Scn8a^med/+ mutant mice. Number of slices = 4-17, number of mice = 2-7. *p < 0.001 when compared to WT; Fisher's exact test with Bonferroni correction.

Figure 2

Analysis of burst characteristics between Scn8a^med/+ and WT mice. A, Hippocampal slices from Scn8a^med/+ mutants demonstrate longer latencies to the onset of burst activity compared to slices from WT mice for all age groups (WT vs mutant at P13-P15: 2.7 ± 0.2 min vs 3.8 ± 0.2 min, p < 0.05; WT vs mutant at P24-P26: 6.1 ± 0.8 min vs 11.1 ± 1.2 min, p < 0.01; WT vs mutant at P70-P84: 6.9 ± 0.9 min vs 9.2 ± 1.2 min, p <0.05). Comparison of burst activity showed similar inter-burst frequency B, and burst duration C, between slices from Scn8a^med/+ mutants and WT mice. P13-P15, n = 4-5; P24-P26, n = 6-9; P70-P84, n = 3-5; Student's two-tailed t test, *p < 0.05; **p < 0.001; error bars indicate SEM.

Figure 3

Developmental expression of VGSCs in the hippocampus of P10-P30 WT mice. A, Western blot analysis of hippocampal membrane fractions from WT mice using rabbit anti-Na_v1.6 polyclonal antibody. Pan-cadherin (135 kDa) was used as a loading control. Statistical analysis of Na_v1.6 levels during development was performed using one-way ANOVA; * p < 0.001. Post hoc analysis demonstrated that Na_v1.6 levels in the hippocampus reach adult levels at 3-4 weeks after birth (p<0.05, Holm-Sidak post hoc, n=3 per age group). Hippocampal tissues from WT and Scn8a^med/+ mice were analyzed by Western blotting (WB) and qPCR for alterations in the expression of B, Scn1a and C, Scn2a. Alpha tubulin (50 kDa) was used a loading control. No significant differences in protein or RNA levels were observed. Scn1a and Scn2a protein and RNA expression levels were analyzed by the Student's two-tailed t test, p>0.05. WB, n = 7 per group; qPCR n = 4-5 per group. Error bars indicate SEM.

Figure 4

Tamoxifen-induced global deletion of the Scn8a gene. A, PCR product amplified from dissected neocortical tissue from flox/+,ER-Cre,TAM mice as well as control littermates (flox/+,ER-Cre,Veh) demonstrated that deletion is only detectable following TAM injection in mice that express the ER-Cre transgene and the floxed Scn8a allele (third lane). Additionally, the Scn8a floxed allele was only marginally detectable following TAM-initiated deletion, providing further evidence of efficient deletion. B, Western blotting demonstrated significant (79%) knockdown of Na_v1.6 protein in homozygous floxed mice carrying the ER-Cre transgene following TAM injection (flox/flox,ER-Cre,TAM) compared to flox/flox,ER-Cre mice administered vehicle. n = 3 per group; Student's two-tailed t test, *p < 0.05; Error bars indicate SEM. C. Representative Western blot from vehicle and TAM treated flox/flox,ER-Cre mice demonstrates reduced band intensity in the TAM treated group.

Figure 5

Inducible deletion of the Scn8a gene in the adult mouse is sufficient to increase the latency to flurothyl- and KA-induced seizures and to protect against electrically induced 6-Hz psychomotor seizures. A, Latencies to flurothyl-induced myoclonic jerk (MJ) and generalized tonic-clonic seizures (GTCS) were increased following TAM-induced inactivation of the Scn8a gene (flox/+,ERCre,TAM) (white bar) compared to control littermates (+/+,no Cre,TAM; +/+,ER-Cre,TAM; flox/+,no Cre,TAM; and flox/+,ER-Cre,Veh) (black bars). n = 10-12 per group. MJ and GTCS, one-way ANOVA, p < 0.001; Dunnett's post hoc, p < 0.05. B, Latencies to KA-induced seizure events were increased following Scn8a deletion (flox/+,ER-Cre,TAM) (solid line) when compared to vehicle-injected controls (flox/+,ER-Cre,Veh) (dashed line). Seizure activity was scored using a modified Racine scale: 1 = freezing, 2 = head nodding, 3 = tail clonus, 4 = forelimb clonus, 5 = rearing and falling, 6 = GTCS. n = 12-13 per group. Two-way ANOVA, p < 0.01; Bonferroni post hoc, p < 0.05. n = 12-13. C, 6-Hz psychomotor seizure severity was decreased after TAM-initiated inactivation of Scn8a (flox/+,ER-Cre,TAM) (solid line) when compared to vehicle-injected controls (flox/+,ER-Cre,Veh) (dashed line). n = 7-9 per group. Mann-Whitney-U test, p < 0.05. Seizure activity was scored using the following modified Racine scale: 1 = freezing, 2 = head nodding 3 = rearing and falling. Average Racine scores: flox/+,ERCre,Veh. pre = 2.1, post = 2.0; flox/+,ER-Cre,TAM pre = 2.0, post = 0.4. D, There was a trend toward reduced 6-Hz psychomotor seizure activity following inactivation of Scn8a (flox/+,ERCre,TAM) when compared to vehicle-injected controls (flox/+,ER-Cre,Veh). n = 7-9 per group; Fisher's exact test, n.s. p = 0.07. *p < 0.05; **p < 0.01; error bars indicate SEM

Figure 6

Lentiviral Cre-mediated knockdown of Scn8a in the hippocampus reduces seizure activity following PT administration. Scn8a^flox/flox mice were injected with either LV-GFP or LV-Cre to achieve knockdown of Scn8a in the hippocampus. A, GFP-positive cells were observed in the CA1, CA3, and DG layers of the hippocampus with highest expression in the DG and CA3. Images were captured at 4X and 40X magnification (inset), scale bar = 100 μm. B, Fewer seizures were recorded in LV-Cre- compared to LV-GFP-treated mice following PT administration. No significant difference in the latency to the first seizure C, or the average duration of each seizure event D, was observed in LV-Cre- compared to LV-GFP-injected mice. E, Representative examples of baseline EEG and seizure activity indicating normal baseline EEG activity in both GFP and Cre groups and seizure activity in Cre and GFP groups following PT administration. n = 6-7 per group. Mann-Whitney-U test, *p < 0.05. Error bars indicate SEM.