Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2007 May 30;27(22):5903-14.
doi: 10.1523/JNEUROSCI.5270-06.2007.

Nav1.1 localizes to axons of parvalbumin-positive inhibitory interneurons: a circuit basis for epileptic seizures in mice carrying an Scn1a gene mutation

Ikuo Ogiwara  1 , Hiroyuki Miyamoto, Noriyuki Morita, Nafiseh Atapour, Emi Mazaki, Ikuyo Inoue, Tamaki Takeuchi, Shigeyoshi Itohara, Yuchio Yanagawa, Kunihiko Obata, Teiichi Furuichi, Takao K Hensch, Kazuhiro Yamakawa
Affiliations
Comparative Study

Nav1.1 localizes to axons of parvalbumin-positive inhibitory interneurons: a circuit basis for epileptic seizures in mice carrying an Scn1a gene mutation

Ikuo Ogiwara et al. J Neurosci. .

Abstract

Loss-of-function mutations in human SCN1A gene encoding Nav1.1 are associated with a severe epileptic disorder known as severe myoclonic epilepsy in infancy. Here, we generated and characterized a knock-in mouse line with a loss-of-function nonsense mutation in the Scn1a gene. Both homozygous and heterozygous knock-in mice developed epileptic seizures within the first postnatal month. Immunohistochemical analyses revealed that, in the developing neocortex, Nav1.1 was clustered predominantly at the axon initial segments of parvalbumin-positive (PV) interneurons. In heterozygous knock-in mice, trains of evoked action potentials in these fast-spiking, inhibitory cells exhibited pronounced spike amplitude decrement late in the burst. Our data indicate that Nav1.1 plays critical roles in the spike output from PV interneurons and, furthermore, that the specifically altered function of these inhibitory circuits may contribute to epileptic seizures in the mice.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Generation of Scn1a knock-in mice with a nonsense mutation. A, The RX nonsense mutation is located within a loop between segments 5 and 6 of domain III and could lead to production of truncated mutant Nav1.1 missing the segment 6 of domain III, domain IV, and C terminus. The position of the RX mutation is represented by an asterisk. B, Schematic representation of the wild-type Scn1a allele, the targeting vector, and the RX mutant Scn1a allele. The nucleotide substitution (CgG to TgA) in exon 21 leading to the RX mutation is represented by an asterisk. The BamHI and PstI sites, the probes used for Southern blot analysis, and the sizes of the restriction fragments detected by the probes are indicated. C, Genotyping of offspring from heterozygote intercrosses by Southern blot analysis of BamHI- or PstI-digested mouse genomic DNA. D, Verification of the presence of the RX mutation using PCR analysis of genomic DNA. The positions of primers and the sizes of PCR products are indicated. The position of the RX mutation is represented by an asterisk.
Figure 2.
Figure 2.
Scn1aRX/RX mice developed spontaneous seizures, were malnourished, and died prematurely. A, Representative EcoG recorded in a P14.5 Scn1a+/+ pup. B, Representative interictal EcoG recorded in a P14.5 Scn1aRX/RX pup. C, D, Two examples of ictal EcoG recorded in two P14.5 Scn1aRX/RX pups. E, Photographs of an Scn1aRX/RX pup (left) and its wild-type littermate (right) at P14.5. F, Body weight of Scn1a+/+ (n = 15), Scn1aRX/+ (n = 40), and Scn1aRX/RX (n = 23) pups at P14.5. Body weight was significantly reduced in Scn1aRX/RX pups compared with their Scn1a+/+ and Scn1aRX/+ littermates (**p < 0.01). Values represent means ± SEM. G, Survival curves of Scn1a+/+ (open circles; n = 26), Scn1aRX/+ (filled rectangles; n = 50), and Scn1aRX/RX (filled triangles; n = 19) mice in a C57BL/6/129 (75%/25%) background from P3. H, Survival curves of Scn1a+/+ (open circles; n = 8), Scn1aRX/+ (filled rectangles; n = 23), and Scn1aRX/RX (filled triangles; n = 8) mice in a C57BL/6/129 (25%/75%) background from P3.
Figure 3.
Figure 3.
Expression of Scn1a mRNA and Nav1.1 protein in P14–P16 Scn1a+/+, Scn1aRX/+, and Scn1aRX/RX pups. A, Northern blot analyses of total brain poly(A) RNA were performed with three different probes (namely, 5′-UTR, coding region, and 3′-UTR probes, respectively). β-Actin was used as internal control. B, Western blot analysis of total brain membrane fraction using the goat anti-internal region Nav1.1 polyclonal antibody (left). This antibody recognized both full-length and truncated mutant Nav1.1 expressed heterologously in HEK293 cells (right). Note that the rabbit anti-internal region Nav1.1 antibody provided an identical pattern (supplemental Fig. 2, available at www.jneurosci.org as supplemental material). β-Tubulin was used as internal control. C, Western blot analysis of total brain membrane fraction using the rabbit anti-C-terminal Nav1.1 polyclonal antibody (left). This antibody recognized full-length Nav1.1 but not truncated mutant Nav1.1 expressed heterologously in HEK293 cells (right). β-Tubulin was used as internal control. D, The developmental expression of Nav1.1 protein in the wild-type mouse brain at different stages. Western blot analysis of total brain membrane fraction was performed with the rabbit anti-C-terminal Nav1.1 polyclonal antibody. β-Tubulin was used as internal control.
Figure 4.
Figure 4.
Regional distribution of Scn1a mRNA and Nav1.1 protein in the developing mouse brain. A, Detection of Scn1a mRNA expression in P14–P16 Scn1a+/+ (a) and Scn1aRX/RX pups (b) by in situ hybridization with the probe complementary to 3′-UTR of Scn1a mRNA. Note that in situ hybridization with another probe complementary to Scn1a mRNA coding region gave an identical pattern (supplemental Fig. 5, available at www.jneurosci.org as supplemental material). Higher-magnified images are also shown in supplemental Figure 6 (available at www.jneurosci.org as supplemental material). Scale bars, 1 mm. B, Detection of Nav1.1 protein in P14–P16 Scn1a+/+ (a) and Scn1aRX/RX (b) pups by immunohistochemistry with the rabbit anti-C-terminal Nav1.1 antibody. Note that the anti-internal region Nav1.1 antibodies provided an identical staining (supplemental Fig. 7A,B, available at www.jneurosci.org as supplemental material). Higher-magnified images are also shown in Figures 5A, 6A, and 7A and supplemental Figure 8A (available at www.jneurosci.org as supplemental material). Scale bars, 1 mm.
Figure 5.
Figure 5.
Nav1.1 localization to AISs of parvalbumin-positive interneurons in the developing neocortex. P14–P16 Scn1a+/+ and Scn1aRX/RX pups were examined. Shown are representative images. Arrowheads indicate examples of Nav1.1-immunoreactive AISs. A, Neocortices of Scn1a+/+ (a) and Scn1aRX/RX (c) pups were stained using the rabbit anti-C-terminal Nav1.1 antibody and DAB. b, d, Higher-magnified images outlined in a and c. Scale bars: a, c, 500 μm; b, d, 40 μm. B, Immunofluorescence histochemistry with the rabbit anti-C-terminal Nav1.1 antibody (a, e; red), together with the anti-βIV-spectrin (b, f; green) and anti-parvalbumin (c, g; blue) antibodies. d, h, Merged images. a–d, Arrows indicate examples of AISs immunopositive for βIV-spectrin but not for Nav1.1. Scale bars, 50 μm. C, Immunofluorescence histochemistry with the rabbit anti-C-terminal Nav1.1 (a; red) and anti-βIV-spectrin (b; green) antibodies, together with 4′-6-diamidino-2-phenylindole (DAPI) (c; blue). d, Merged images. Arrows indicate examples of AISs immunopositive for βIV-spectrin but not for Nav1.1. Scale bars, 10 μm. D, Immunofluorescence histochemistry with the rabbit anti-C-terminal Nav1.1 antibody (a, d; red), together with the anti-phosphorylated neurofilament antibody mixture (SMI312) (b, e; green). c, f, Merged images. Scale bars, 50 μm. All images are oriented from pial surface (top) to callosal (bottom).
Figure 6.
Figure 6.
Nav1.1 localization to somata and axons of parvalbumin-positive interneurons in the developing hippocampus. P14–P16 Scn1a+/+ and Scn1aRX/RX pups were examined. Shown are representative images. Arrowheads and arrows indicate examples of Nav1.1-immunoreactive fibers and somata, respectively. A, Hippocampi of Scn1a+/+ (a) and Scn1aRX/RX (c) pups were stained using the rabbit anti-C-terminal Nav1.1 antibody and DAB. b, d, Higher-magnified images outlined in a and c. Scale bars: a, c, 500 μm; b, d, 40 μm. B, Immunofluorescence histochemistry with the rabbit anti-C-terminal Nav1.1 antibody (a, e, i, m; red), together with the anti-βIV-spectrin (b, f, j, n; green) and anti-parvalbumin (c, g, k, o; blue) antibodies. d, h, l, p, Merged images. Scale bars, 50 μm. C, Immunofluorescence histochemistry with the rabbit anti-C-terminal Nav1.1 antibody (a, d; red), together with the anti-phosphorylated neurofilament antibody mixture (SMI312) (b, e; green). c, f, Merged images. Scale bars, 50 μm. DG, Dentate gyrus; O, stratum oriens; P, stratum pyramidale; R, stratum radiatum.
Figure 7.
Figure 7.
Nav1.1 localization to axons in the developing cerebellar cortex. P14–P16 Scn1a+/+ and Scn1aRX/RX pups were examined. Shown are representative images. Arrowheads indicate examples of Nav1.1-immunoreactive fibers. A, Cerebellar cortices of Scn1a+/+ (a) and Scn1aRX/RX (c) pups were stained using the rabbit anti-C-terminal Nav1.1 antibody and DAB. b, d, Higher-magnified images outlined in a and c. Scale bars: a, c, 500 μm; b, d, 40 μm. B, Immunofluorescence histochemistry with the rabbit anti-C-terminal Nav1.1 antibody (a, e, i; red), together with the anti-βIV-spectrin (b, f, j; green) and anti-parvalbumin (c, g, k; blue) antibodies. d, h, l, Merged images. e–h, Double arrowheads indicate examples of Nav1.1-immunoreactive AISs of Purkinje cells. Scale bars, 50 μm. C, Immunofluorescence histochemistry with the rabbit anti-C-terminal Nav1.1 antibody (a, d; red), together with the anti-phosphorylated neurofilament antibody mixture (SMI312) (b, e; green). c, f, Merged images. Scale bars, 50 μm. IC, Inferior colliculus; CL, cerebellar lobule; M, molecular cell layer; P, Purkinje cell layer; G, granule cell layer.
Figure 8.
Figure 8.
Summary of electrophysiological properties of Gad1GFP/+:Scn1a+/+ (WT; open circles; n = 8 neurons) and Gad1GFP/+:Scn1aRX/+ (HET; filled circles; n = 8 neurons) interneurons. A, Properties (half-width and spike amplitude) of single action potentials at threshold and spike decrement of interneurons to a prolonged depolarizing current pulse (5× threshold; 500 ms). Spike decrement was calculated as percentage of last spike amplitude divided by first spike amplitude. Values represent means ± SEM; **p < 0.01. B, Representative traces of a WT (top) and HET (bottom) interneuron to a depolarizing current pulse at 5× threshold. Note that the rate of spike firing decreases mainly in HET interneurons. Representative biocytin-filled interneurons are shown for WT and HET mice. Scale bars, 50 μm. C, Membrane properties of interneurons. Values represent means ± SEM; *p < 0.05. D, Plot of the current–voltage relationship of interneurons in WT (broken line) and HET (straight line) mice. Points represent voltages recorded at different injected current pulses. Linear regression is shown for each case.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bacci A, Huguenard JR. Enhancement of spike-timing precision by autaptic transmission in neocortical inhibitory interneurons. Neuron. 2006;49:119–130. - PubMed
    1. Beckh S, Noda M, Lubbert H, Numa S. Differential regulation of three sodium channel messenger RNAs in the rat central nervous system during development. EMBO J. 1989;8:3611–3616. - PMC - PubMed
    1. Berghs S, Aggujaro D, Dirkx R, Jr, Maksimova E, Stabach P, Hermel JM, Zhang JP, Philbrick W, Slepnev V, Ort T, Solimena M. βIV spectrin, a new spectrin localized at axon initial segments and nodes of Ranvier in the central and peripheral nervous system. J Cell Biol. 2000;151:985–1002. - PMC - PubMed
    1. Bishop GA. An analysis of HRP-filled basket cell axons in the cat's cerebellum. I. Morphometry and configuration. Anat Embryol (Berl) 1993;188:287–297. - PubMed
    1. Black JA, Yokoyama S, Higashida H, Ransom BR, Waxman SG. Sodium channel mRNAs I, II and III in the CNS: cell-specific expression. Brain Res Mol Brain Res. 1994;22:275–289. - PubMed

Publication types

MeSH terms