Elsevier

Neurobiology of Disease

Volume 49, January 2013, Pages 211-220
Neurobiology of Disease

Preferential inactivation of Scn1a in parvalbumin interneurons increases seizure susceptibility

https://doi.org/10.1016/j.nbd.2012.08.012Get rights and content

Abstract

Voltage-gated sodium channels (VGSCs) are essential for the generation and propagation of action potentials in electrically excitable cells. Dominant mutations in SCN1A, which encodes the Nav1.1 VGSC α-subunit, underlie several forms of epilepsy, including Dravet syndrome (DS) and genetic epilepsy with febrile seizures plus (GEFS +). Electrophysiological analyses of DS and GEFS + mouse models have led to the hypothesis that SCN1A mutations reduce the excitability of inhibitory cortical and hippocampal interneurons. To more directly examine the relative contribution of inhibitory interneurons and excitatory pyramidal cells to SCN1A-derived epilepsy, we first compared the expression of Nav1.1 in inhibitory parvalbumin (PV) interneurons and excitatory neurons from P22 mice using fluorescent immunohistochemistry. In the hippocampus and neocortex, 69% of Nav1.1 immunoreactive neurons were also positive for PV. In contrast, 13% and 5% of Nav1.1 positive cells in the hippocampus and neocortex, respectively, were found to co-localize with excitatory cells identified by CaMK2α immunoreactivity. Next, we reduced the expression of Scn1a in either a subset of interneurons (mainly PV interneurons) or excitatory cells by crossing mice heterozygous for a floxed Scn1a allele to either the Ppp1r2-Cre or EMX1-Cre transgenic lines, respectively. The inactivation of one Scn1a allele in interneurons of the neocortex and hippocampus was sufficient to reduce thresholds to flurothyl- and hyperthermia-induced seizures, whereas thresholds were unaltered following inactivation in excitatory cells. Reduced interneuron Scn1a expression also resulted in the generation of spontaneous seizures. These findings provide direct evidence for an important role of PV interneurons in the pathogenesis of Scn1a-derived epilepsies.

Highlights

► Nav1.1 is expressed in most PV interneurons in the neocortex and hippocampus. ► Nav1.1 is expressed in 5–13% of excitatory cells in neocortex and hippocampus. ► Preferential inactivation of one Scn1a allele in PV interneurons reduces seizure thresholds. ► Inactivation of one Scn1a allele in excitatory cells does not alter seizure thresholds. ► Preferential inactivation of one Scn1a allele in PV interneurons results in spontaneous seizures.

Section snippets

Preparation of the targeting construct

The targeting construct was generated by cloning three PCR-generated fragments with homology to the Scn1a locus into the pFlexible vector (pLoxP-2FRT-PGKneo) (van der Weyden et al., 2005). Fragment 1 (3.8 kb), corresponding to the 5′ arm of homology, was amplified using the primer pair 1F/1R. Fragment 2 (750 bp), containing exon 1 and flanking non-coding sequences, was amplified using the primer pair 2F/2R. Fragment 3 (4.1 kb), the 3′ arm of homology, was amplified using the primer pair 3F/3R (

Generation of the floxed Scn1a allele

We generated an Scn1a conditional knock-out mouse model in which exon 1 of the mouse Scn1a gene is flanked by LoxP sites (Fig. 1A). Five hundred and seventy six neomycin-resistant ES cell clones were screened for correct targeting by PCR amplification of the 5′ and 3′ regions of homology. Clones that were positive in both PCR assays were also examined by Southern blot analysis. Two correctly targeted ES cell clones were injected into blastocysts to generate chimeric mice that were bred to

Discussion

In the current study we generated an Scn1a conditional knockout line that, following global deletion of Scn1a, recapitulated the reduced lifespan and spontaneous seizures observed in the Scn1a knock-out mouse (a model of DS) and the R1648H GEFS + mouse model (Martin et al., 2010, Yu et al., 2006). The conditional knockout line was then used to determine the effect of preferentially inactivating Nav1.1 from PV interneurons and excitatory cells. We found that preferential inactivation of one Scn1a

Conclusions

In summary, we found that preferential inactivation of one Scn1a allele in PV interneurons of the neocortex and hippocampus results in reduced seizure thresholds, whereas similar inactivation of Scn1a from excitatory neurons does not significantly affect seizure thresholds. This work represents the first direct demonstration of a causal relationship between loss of Nav1.1 in a particular neuronal subtype and seizure susceptibility. The identification of critical neuronal subtypes that

Acknowledgments

We would like to thank the laboratory of Dr. William Catterall for assistance with the hyperthermia seizure induction paradigm and guidance with immunohistochemistry. We are grateful to Cheryl Strauss for editorial assistance. This study was supported by grants from the NIH (R01 NS072221 to A.E., and 1F31 NS065694 to S.B.D.) and by a predoctoral fellowship from the Epilepsy Foundation (S.B.D.). This research was also supported in part by the NINDS core facilities grant P30N5055077 to the Emory

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