Elsevier

Neurobiology of Disease

Volume 65, May 2014, Pages 1-11
Neurobiology of Disease

Strain- and age-dependent hippocampal neuron sodium currents correlate with epilepsy severity in Dravet syndrome mice

https://doi.org/10.1016/j.nbd.2014.01.006Get rights and content

Highlights

  • Scn1a+/− mice have a more severe phenotype on the (129xB6)F1 strain compared to 129.

  • Interneuron sodium current density is reduced in affected F1.Scn1a+/− mice.

  • Interneuron sodium current density is unchanged in unaffected 129.Scn1a+/− mice.

  • Scn1a+/− pyramidal neurons exhibit age-dependent elevation of sodium current density.

  • Elevation of pyramidal neuron sodium current density correlates with premature lethality peak.

Abstract

Heterozygous loss-of-function SCN1A mutations cause Dravet syndrome, an epileptic encephalopathy of infancy that exhibits variable clinical severity. We utilized a heterozygous Scn1a knockout (Scn1a+/−) mouse model of Dravet syndrome to investigate the basis for phenotype variability. These animals exhibit strain-dependent seizure severity and survival. Scn1a+/− mice on strain 129S6/SvEvTac (129.Scn1a+/−) have no overt phenotype and normal survival compared with Scn1a+/− mice bred to C57BL/6J (F1.Scn1a+/−) that have severe epilepsy and premature lethality. We tested the hypothesis that strain differences in sodium current (INa) density in hippocampal neurons contribute to these divergent phenotypes. Whole-cell voltage-clamp recording was performed on acutely-dissociated hippocampal neurons from postnatal days 21–24 (P21–24) 129.Scn1a+/− or F1.Scn1a+/− mice and wild-type littermates. INa density was lower in GABAergic interneurons from F1.Scn1a+/− mice compared to wild-type littermates, while on the 129 strain there was no difference in GABAergic interneuron INa density between 129.Scn1a+/− mice and wild-type littermate controls. By contrast, INa density was elevated in pyramidal neurons from both 129.Scn1a+/− and F1.Scn1a+/− mice, and was correlated with more frequent spontaneous action potential firing in these neurons, as well as more sustained firing in F1.Scn1a+/− neurons. We also observed age-dependent differences in pyramidal neuron INa density between wild-type and Scn1a+/− animals. We conclude that preserved INa density in GABAergic interneurons contributes to the milder phenotype of 129.Scn1a+/− mice. Furthermore, elevated INa density in excitatory pyramidal neurons at P21–24 correlates with age-dependent onset of lethality in F1.Scn1a+/− mice. Our findings illustrate differences in hippocampal neurons that may underlie strain- and age-dependent phenotype severity in a Dravet syndrome mouse model, and emphasize a contribution of pyramidal neuron excitability.

Introduction

Idiopathic epilepsies are a group of clinically diverse disorders with a strong genetic component to their pathogenesis. Although most epilepsies exhibit complex inheritance, some result from single gene mutations. Mutations in genes encoding neuronal voltage-gated sodium channels (NaV) result in genetic epilepsies with overlapping clinical characteristics but divergent clinical severity (Meisler and Kearney, 2005). Mutation of SCN1A, encoding the pore-forming subunit NaV1.1, is the most commonly discovered cause of monogenic epilepsies (Catterall et al., 2010). More than 800 heterozygous SCN1A epilepsy-associated mutations have been identified, with more than 70% occurring in patients with Dravet syndrome (DS), also known as severe myoclonic epilepsy of infancy (Claes et al., 2009, Lossin, 2009). While DS is typically characterized by seizure onset in the first year of life with an ensuing epileptic encephalopathy consisting of cognitive, behavioral, and motor impairments, the severity of its presentation and progression can be variable (Brunklaus et al., 2012, Zuberi et al., 2011). However, it remains unclear why individuals bearing the same heterozygous SCN1A mutation exhibit divergent seizure phenotypes, even within the same family (Goldberg-Stern et al., 2013, Kimura et al., 2005, Pineda-Trujillo et al., 2005).

Genetic modifiers may contribute to the variable expressivity of SCN1A mutations in DS patients and this notion is further suggested by investigations of Scn1a knockout (Scn1a+/−) and Scn1a-R1407X knock-in mouse models of DS (Miller et al., 2013, Ogiwara et al., 2007, Yu et al., 2006). Heterozygous DS mice display spontaneous seizures and premature death as well as cognitive and motor impairments (Han et al., 2012, Ito et al., 2012, Kimura et al., 2005). Reduced sodium current (INa) density in morphologically identified hippocampal interneurons correlated with impaired excitability, whereas INa density was not different in excitatory pyramidal neurons (Bechi et al., 2012, Yu et al., 2006). These studies suggested that dysfunctional inhibitory circuits may underlie the pathophysiology of DS. Importantly, the phenotype severity observed in Scn1a+/− mice is influenced by genetic background. Scn1a+/− mice maintained on the 129 strain background have a normal lifespan with no seizures. By contrast, crossing 129.Scn1a+/− mice to C57BL/6 animals generates offspring with overt seizures and decreased lifespan (Miller et al., 2013, Yu et al., 2006). The neurophysiological basis of strain-dependent seizure severity observed in Scn1a+/− mice has not been investigated, but represents an opportunity to elucidate neuronal mechanisms responsible for variable disease expression.

In this study, we compared the properties of INa in hippocampal neurons from Scn1a+/− mice on different genetic backgrounds in order to test the hypothesis that variable sodium current compensation in hippocampal neurons accounts for strain-dependent phenotype differences. We observed significant differences in both interneuron and pyramidal neuron INa densities that correlated with strain- and age-dependent phenotypes. Our findings contribute a plausible explanation for the divergent seizure phenotypes observed in DS and offer new opportunities to connect genetic modifiers with neurophysiological mechanisms with relevance to epileptogenesis.

Section snippets

Generation of Scn1a+/− mice

The Scn1atm1Kea targeted null allele was generated by homologous recombination in TL1 ES cells (129S6/SvEvTac). Exon 1 of the mouse Scn1a gene was replaced by a selection cassette as described (Miller et al., 2013). The resultant Scn1a+/− mouse line (129.Scn1a+/−) was then maintained on the 129S6/SvEvTac (129) inbred strain by continuous backcrossing to 129. Strain C57BL/6J (B6) was crossed to 129.Scn1a+/− to generate (129.Scn1a+/− x B6)F1 offspring (designated as F1.Scn1a+/−) for experiments.

Strain-dependent seizure severity and survival of Scn1a+/− mice

Heterozygous F1.Scn1a+/− mice exhibited spontaneous seizures beginning at P18 and premature lethality, similar to the phenotypes reported for the Scn1a exon 26 knockout and Scn1a-R1407X knock-in heterozygotes (Miller et al., 2013, Ogiwara et al., 2007, Yu et al., 2006). However, heterozygous 129.Scn1a+/− mice did not exhibit any overt phenotype (Miller et al., 2013).

Lifespan was significantly influenced by strain background with F1.Scn1a+/− mice exhibiting substantially reduced survival

Discussion

Genetic epilepsies caused by mutations in genes encoding voltage-gated sodium channels exhibit variable expressivity, a common phenomenon among monogenic disorders and often ascribed to the action of genetic modifiers. Strain-dependence of phenotype severity in genetically engineered mice can provide a tractable model for investigating the genetic basis of variable disease expression (Kearney, 2011). In this study, we exploited a DS mouse model to investigate the neurophysiological basis for

Conclusions

We have shown that strain-dependent epilepsy observed in Scn1a+/− mice on F1 and 129 strain backgrounds results from a combination of a loss-of-function phenotype in inhibitory interneurons and a gain-of-function phenotype in excitatory pyramidal cells. We conclude that age-dependent changes in sodium channel currents contribute to variable excitability profiles of these cells and strain-dependent seizure phenotypes.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Abbreviations

    129

    129S6/SvEvTac

    B6

    C57BL/6J

    DS

    Dravet syndrome

    EEG

    electroencephalography

    EPSC

    excitatory post-synaptic current

    Flurothyl

    2,2,2-trifluroethylether

    GNa

    sodium whole-cell conductance

    INa

    sodium current

    M-MLV

    Moloney Murine Leukemia Virus

    NaV

    voltage-gated sodium channel

    TTX

    tetrodotoxin

Acknowledgments

We thank Andrew Tapper, Ph.D., for providing PCR primer sequences of Gad67 gene, Clint McCollom for mouse husbandry, Jennifer Kunic for TaqMan probe and primer design, and Danny Winder for assistance with current clamp experimental design and analysis. This work was supported by National Institutes of Health grants [NS032387 to A.L.G., NS053792, NS063097 to J.A.K.]; and Howard Hughes Medical Institute Medical Research Fellowship to A.M.M.

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