Generation of a floxed Scn1a allele in mice. (A) Verification of floxed and deleted exon 7 alleles using PCR analysis of genomic DNA. Primer positions and PCR product sizes are indicated. (B) Western blot analysis of brain membrane proteins prepared from 5-month-old wild-type and homozygous floxed Scn1a (Scn1a^fl/fl) mice, using anti-Nav1.1 antibody. β-Tubulin was used as an internal control, and two independent assays were performed. (C) Western blot analysis of brain membrane proteins prepared from P14.5 Scn1a^+/+, Scn1a^d/+ and Scn1a^d/d littermates, using anti-Nav1.1 antibody. β-Tubulin was used as an internal control, and two independent assays were performed. (D and E) Representative interictal (D) and ictal (E) ECoG recordings from Scn1a^d/d mice. ECoG recordings were performed with P14–16 Scn1a^d/d mice and Scn1a^+/+ controls (n = 3, each group). (F) Survival curves of P3 Scn1a^+/+, Scn1a^d/+ and Scn1a^d/d littermates. +, wild-type allele; fl, floxed allele; d, deleted allele.

Generation of VGAT-Cre transgenic mice. (A) Schematic representation of the DNA construct used to generate the VGAT-Cre transgenic mouse line. (B) A representative parasagittal section from 8-week-old Rosa26-LacZ,VGAT-Cre brain, stained with anti-β-galactosidase antibody. Scale bar: 300 µm. (C–J) Representative images of immunofluorescence histochemistry in the hippocampus (C–F) and neocortical layer II/III (G–J) in 8-week-old Rosa26-LacZ,VGAT-Cre mice, stained with anti-β-galactosidase antibody (C and G; green), anti-GABA antibody (D and H; red) and DAPI (E and I; blue), with respective merged images (F and J). Arrowheads indicate cells double-labeled with β-galactosidase and GABA. Note that cells immunopositive for β-galactosidase (indicative of Cre-loxP recombination in the Rosa26-LacZ allele) were usually immunopositive for GABA also. o, stratum oriens; p, stratum pyramidale; r, stratum radiatum. Scale bars: 50 µm.

Selective Nav1.1 deletion in mouse inhibitory neurons, using VGAT-Cre results in severe epileptic seizures and high lethality. (A) Verification of VGAT-Cre-dependent Scn1a gene deletion in the floxed allele in P21.5 Scn1a^fl/+,VGAT-Cre brain. PCR analysis of brain genomic DNA isolated from P21.5 Scn1a^fl/+, VGAT-Cre and Scn1a^fl/fl mice was performed. (B and C) Verification of VGAT-Cre-dependent Nav1.1 deletion by semi-quantitative western blot analysis of brain membrane proteins prepared from P21.5 Scn1a^fl/+, VGAT-Cre mice and Scn1a^fl/fl littermates, using anti-Nav1.1 antibody (B). β-Tubulin was used as an internal control. Mean Nav1.1 expression levels in Scn1a^fl/+, VGAT-Cre mice are represented as percentages, relative to Nav1.1 in Scn1a^fl/+ littermates (100%; C). (D and E) Representative ECoG of a lethal seizure in a P18–21 Scn1a^fl/+,VGAT-Cre mouse. An arrow indicates onset of the epileptiform discharge. ECoG recordings were performed with P18–21 Scn1a^fl/+,VGAT-Cre and Scn1a^fl/+ control mice (n = 3, each group). (F) Survival curves of P3 Scn1a^fl/+, Scn1a^d/+ (reprinted from Fig. F for comparison), Scn1a^fl/+,VGAT-Cre and Scn1a^fl/fl,VGAT-Cre mice. Note that all Scn1a^fl/fl,VGAT-Cre mice died before P15, and that all but one Scn1a^fl/+,VGAT-Cre mice died before P35. (G) Verification of VGAT-Cre-dependent Scn1a gene deletion in the floxed allele in P12.5 Scn1a^fl/fl,VGAT-Cre brain by PCR analysis. (H and I) Verification of VGAT-Cre-dependent Nav1.1 deletion by semi-quantitative western blot analysis of brain membrane proteins prepared from P12.5 Scn1a^fl/fl, VGAT-Cre mice and age-matched Scn1a^fl/fl controls, using anti-Nav1.1 antibody. Two independent assays were performed. Error bars represent SEM. *P < 0.05, **P < 0.01. +, wild-type allele; fl, floxed allele; d, deleted allele.

Nav1.1 immunosignals are significantly reduced in inhibitory neurons of Scn1a^fl/fl,VGAT-Cre mice. (A–D) Representative parasagittal sections of P12.5 Scn1a^fl/fl (A) and Scn1a^fl/fl,VGAT-Cre (B) hippocampi, and P12.5 Scn1a^fl/fl (C) and Scn1a^fl/fl,VGAT-Cre (D) neocortices, stained with anti-Nav1.1 antibody. Higher magnification images outlined in (a) are shown in (b). An arrowhead indicates an Nav1.1-immunoreactive proximal neurite oriented toward pial surface, which putatively corresponds to an AIS of a PV cell (,) (see also Fig. O). Scale bars: (a) 400 µm; (b) 40 µm. DG, dentate gyrus; o, stratum oriens; p, stratum pyramidale; l, stratum lucidum. (E and F) Representative parasagittal sections of P12.5 Scn1a^fl/fl (E) and Scn1a^fl/fl, VGAT-Cre (F) cerebellum stained with anti-Nav1.1 antibody. Higher magnification images outlined in (a) are shown in (b) and (d). Higher magnification images outlined in (b) are shown in (c). Arrowheads indicate putative Nav1.1-immunoreactive AISs in Purkinje cells. Arrows indicate Nav1.1-immunoreactive axons of basket cells. Scale bars: (a) 400 µm; (b and c) 40 µm; (d) 80 µm. M, molecular cell layer; P, Purkinje cell layer; G, granule cell layer; CbN, cerebellar nuclei. Nuclear immunosignals are non-specific (). Images are oriented from the pial surface (top) to the callosal (bottom), and from rostral (left) to caudal (right). fl, floxed allele.

Selective Nav1.1 deletion in forebrain excitatory neurons by Emx1-Cre recombination did not cause any observable abnormality in mice. (A) Verification of Emx1-Cre-dependent deletion of the floxed Scn1a gene in the 8-week-old Scn1a^fl/fl, Emx1-Cre brain by PCR analysis. (B and C) Verification of Emx1-Cre-dependent Nav1.1 deletion by semi-quantitative western blot analysis of brain membrane proteins prepared from P21.5 and 8-week-old Scn1a^fl/fl, Emx1-Cre and Scn1a^fl/fl littermates, using anti-Nav1.1 antibody. (D) Survival curves of P3 Scn1a^fl/fl and Scn1a^fl/fl, Emx1-Cre littermates. In both genotypes, a few mice died of unknown reasons. +, wild-type allele; fl, floxed allele. *P < 0.05, ***P < 0.001.

Nav1.1 immunosignals in forebrain excitatory neurons are reduced in Scn1a^fl/fl, Emx1-Cre mice. (A and B) Representative parasagittal sections of P21.5 Scn1a^fl/fl (A) and Scn1a^fl/fl, Emx1-Cre (B) hippocampi stained with anti-Nav1.1 antibody. Higher magnification images outlined in (a) are shown in (b). Scale bars: (a) 400 µm; (b) 40 µm. DG, dentate gyrus; o, stratum oriens; p, stratum pyramidale; l, stratum lucidum. (C and D) Representative parasagittal sections of P21.5 Scn1a^fl/fl (C) and Scn1a^fl/fl, Emx1-Cre (D) neocortices, stained with anti-Nav1.1 antibody. Higher magnification images outlined in (a) are shown in (b). Scale bars: (a) 400 µm; (b) 40 µm. Arrows indicate Nav1.1-immunoreactive neurites oriented toward the pial surface, which putatively correspond to AISs of neocortical PV cells (,) (see also Fig. O). Double arrowheads indicate Nav1.1-immunoreactive proximal neurites oriented toward the ventricular surface in neocortical layer V. (E–H) Representative parasagittal sections of 8-week-old Scn1a^fl/fl (E) and Scn1a^fl/fl, Emx1-Cre (F) hippocampi, and 8-week-old Scn1a^fl/fl (G) and Scn1a^fl/fl, Emx1-Cre (H) neocortices, stained with anti-Nav1.1 antibody. Higher magnification images outlined in (a) are shown in (b). Scale bars: (a) 400 µm; (b) 40 µm. Arrowheads indicate the Nav1.1-immunoreactive bands extending from the stratum lacunosum-moleculare within the CA fields to the molecular layer of dentate gyrus, which virtually correspond to the hippocampal perforant path. DG, dentate gyrus; o, stratum oriens; p, stratum pyramidale; l, stratum lucidum; r, stratum radiatum; lm, stratum lacunosum-moleculare; m, dentate gyrus molecular layer; g, dentate gyrus granule cell layer. (I and J) Representative coronal sections of 8-week-old Scn1a^fl/fl (I) and Scn1a^fl/fl, Emx1-Cre (J) brains stained with anti-Nav1.1 antibody (n = 3 per genotype). Arrowheads and arrows indicate the Nav1.1-immunoreactive bands, which correspond to hippocampal perforant path and corpus callosum, respectively. Higher magnification images outlined in (a) are shown in (b). Note that reductions in Nav1.1 immunostaining intensities in the medial perforant pathway, corpus callosum and neocortical layers II/III, V and VI in Scn1a^fl/fl, Emx1-Cre mice were observed in comparison with Scn1a^fl/fl controls. CC, corpus callosum; DG, dentate gyrus; S1BF, primary somatosensory cortex, barrel field. Scale bars: 100 µm. Nuclear immunosignals are non-specific (). Parasagittal images are oriented from the pial surface (top) to the callosal (bottom), and from rostral (left) to caudal (right). Coronal images are oriented from lateral (left) to medial (right). fl, floxed allele.

Additional Emx1-Cre-mediated Nav1.1 deletion in Scn1a^fl/+, VGAT-Cre mice ameliorates seizure-related sudden death. Survival curves of P3 Scn1a^fl/+, Scn1a^fl/+, Emx1-Cre, Scn1a^fl/+, Emx1-Cre,VGAT-Cre and Scn1a^fl/+, VGAT-Cre littermates. Survival curve of Scn1a^d/+ (reprinted from Fig. F) is included for comparison. Note that a significant decrease in premature lethality in Scn1a^fl/+, Emx1-Cre,VGAT-Cre mice was observed in comparison with Scn1a^fl/+, VGAT-Cre mice. +, wild-type allele; fl, floxed allele.

Selective Nav1.1 deletion in mouse PV cells by PV-Cre causes spontaneous epileptic seizures and premature death. (A) Verification of PV-Cre-TG-dependent floxed Scn1a gene deletion in the brain by PCR analysis. (B and C) Verification of PV-Cre-TG-dependent Nav1.1 deletion by semi-quantitative western blot analysis on brain membrane proteins prepared from P21.5 Scn1a^fl/fl,PV-Cre-TG and Scn1a^fl/fl littermates, using anti-Nav1.1 antibody. Error bars represent SEM; two independent assays were performed. (D and E) Representative interictal (D) and ictal (E) ECoG recordings in P14–16 Scn1a^fl/fl, PV-Cre-TG mice and Scn1a^fl/fl control mice (n = 3, each group). (F) Survival curves of P3 Scn1a^fl/fl, Scn1a^fl/+, Scn1a^fl/+, PV-Cre-TG and Scn1a^fl/fl,PV-Cre-TG littermates. Note that all Scn1a^fl/fl, PV-Cre-TG mice died before P25. +, wild-type allele; fl, floxed allele. ***P < 0.001.

Nav1.1 immunosignals in PV inhibitory neurons are reduced at later developmental stages in Scn1a^fl/fl, PV-TG-Cre mice. (A–N) Representative parasagittal sections of P12.5 Scn1a^fl/fl (A) and Scn1a^fl/fl, PV-Cre-TG (B) hippocampi, P12.5 Scn1a^fl/fl (C) and Scn1a^fl/fl, PV-Cre-TG (D) neocortices, P16.5 Scn1a^fl/fl (E) and Scn1a^fl/fl, PV-Cre-TG (F) hippocampi, P16.5 Scn1a^fl/fl (G) and Scn1a^fl/fl, PV-Cre-TG (H) neocortices, P21.5 Scn1a^fl/fl (I) and Scn1a^fl/fl, PV-Cre-TG (J) hippocampi and P21.5 Scn1a^fl/fl (K and M) and Scn1a^fl/fl, PV-Cre-TG (L and N) neocortices, stained with anti-Nav1.1 antibody. P21.5 was the oldest age used for analysis as Scn1a^fl/fl, PV-TG-Cre mice show a high mortality rate beyond P15 and a life expectancy within P30. Higher magnification images outlined in (a) are shown in (b). Arrowheads indicate putative Nav1.1-immunoreactive AISs of neocortical PV cells. Double arrowheads indicate Nav1.1-immunoreactive proximal neurites oriented toward the ventricular surface. Scale bars: (a) 400 µm; (b) 40 µm. DG, dentate gyrus; o, stratum oriens; p, stratum pyramidale; l, stratum lucidum. (O and P) Immunofluorescence histochemistry of neocortices of P21.5 Scn1a^fl/fl (O) and Scn1a^fl/fl, PV-TG-Cre (P) mice stained with anti-Nav1.1 (a; red), PV (b; green) and ankyrin G (c; cyan) antibodies and counterstained with DAPI (d; blue). Merged images are shown (e). Arrowheads indicate AISs of neocortical PV cells with strong Nav1.1 immunofluorescence. An arrow indicates an AIS of a PV cell with reduced Nav1.1 immunofluorescence. Scale bars: 100 µm. (Q and R) Representative parasagittal sections of P12.5 Scn1a^fl/fl (Q) and Scn1a^fl/fl, PV-TG-Cre (R) cerebellum stained with anti-Nav1.1 antibody. Higher magnification images outlined in (a) are shown in (b) and (d). Higher magnification images outlined in (b) are shown in (c). Scale bars: (a) 400 µm; (b and c) 40 µm; (d) 80 µm. Arrowheads and arrows indicate Nav1.1-immunoreactive AISs of Purkinje cells and axons of basket cells, respectively. M, molecular cell layer; P, Purkinje cell layer; G, granule cell layer; CbN, cerebellar nuclei. Nuclear immunosignals are non-specific (). Images are oriented from the pial surface (top) to the callosal (bottom), and from rostral (left) to caudal (right). fl, floxed allele.

Minimal selective Nav1.1 deletion in PV cells by low-efficiency PV-Cre recombination is sufficient to cause spontaneous epileptic seizures and occasional premature death in mice. (A) Verification of PV-Cre-KI-dependent floxed Scn1a gene deletion in Scn1a^fl/fl, PV-Cre-KI brain by PCR analysis. (B and C) Verification of PV-Cre-KI-dependent Nav1.1 deletion by semi-quantitative western blot analysis of brain membrane proteins prepared from P21.5 and 8-week-old Scn1a^fl/fl, PV-Cre-KI and Scn1a^fl/fl littermates, using anti-Nav1.1 antibody (B). Mean Nav1.1 expression levels in Scn1a^fl/fl, PV-Cre-KI mice are represented as percentages relative to Scn1a^fl/fl mice (100%) (C). Error bars represent SEM; two independent assays were performed. (D–F) Spontaneous epileptic seizures in Scn1a^fl/fl, PV-Cre-KI mice. Representative normal interictal (D), ictal (E) and abnormal interictal (F) ECoGs recorded in Scn1a^fl/fl, PV-Cre-KI mice are shown. ECoG recordings were performed with P25–30 Scn1a^fl/fl, PV-Cre-KI and Scn1a^fl/fl control mice. (G) Survival curves of P3 Scn1a^fl/fl, Scn1a^fl/+, Scn1a^fl/+, PV-Cre-KI and Scn1a^fl/fl, PV-Cre-KI littermates. A proportion of Scn1a^fl/fl, PV-Cre-KI mice suffered sporadic death after P25. +, wild-type allele; fl, floxed allele.

Nav1.1 immunosignals in PV inhibitory neurons are moderately reduced at later developmental stages in Scn1a^fl/fl, PV-KI-Cre mice. (A–L) Representative parasagittal sections of P16.5 Scn1a^fl/fl (A) and Scn1a^fl/fl, PV-Cre-KI (B) hippocampi, P16.5 Scn1a^fl/fl (C) and Scn1a^fl/fl, PV-Cre-KI (D) neocortices, P21.5 Scn1a^fl/fl (E) and Scn1a^fl/fl, PV-Cre-KI (F) hippocampi, P21.5 Scn1a^fl/fl (G) and Scn1a^fl/fl, PV-Cre-KI (H) neocortices, 8-week-old Scn1a^fl/fl (I) and Scn1a^fl/fl, PV-Cre-KI (J) hippocampi and 8-week-old Scn1a^fl/fl (K) and Scn1a^fl/fl, PV-Cre-KI (L) neocortices, stained with anti-Nav1.1 antibody. Higher magnification images outlined in (a) are shown in (b). Scale bars: (a) 400 µm; (b) 40 µm. Arrowheads and double arrowheads indicate Nav1.1-immunoreactive AISs of neocortical PV cells and hippocampal perforant path afferents, respectively. DG, dentate gyrus; o, stratum oriens; p, stratum pyramidale; l, stratum lucidum; r, stratum radiatum; lm, stratum lacunosum-moleculare; m, dentate gyrus molecular layer; g, dentate gyrus granule cell layer. (M and N) Representative parasagittal sections of P21.5 Scn1a^fl/fl (M) and Scn1a^fl/fl, PV-Cre-KI (N) cerebellum, stained with anti-Nav1.1 antibody. Higher magnification images outlined in (a) are shown in (b) and (d). Higher magnification images outlined in (b) are shown in (c). Arrows indicate putative Nav1.1-immunoreactive axons of basket cells. Scale bars: (a) 400 µm; (b and c) 40 µm; (d) 80 µm. M, molecular cell layer; P, Purkinje cell layer; G, granule cell layer; CbN, cerebellar nuclei. Nuclear Nav1.1 immunostaining is non-specific (). Images are oriented from the pial surface (top) to the callosal (bottom), and from rostral (left) to caudal (right). fl, floxed allele.