Strain- and age-dependent hippocampal neuron sodium currents correlate with epilepsy severity in Dravet syndrome mice
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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These authors contributed equally to this work.