Preferential inactivation of Scn1a in parvalbumin interneurons increases seizure susceptibility
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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