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Scn1b deletion in adult mice results in seizures and SUDEP

Abstract Pathogenic loss‐of‐function variants in SCN1B are linked to Dravet syndrome (DS). Previous work suggested that neuronal pathfinding defects underlie epileptogenesis and SUDEP in the Scn1b null mouse model of DS. We tested this hypothesis by inducing Scn1b deletion in adult mice that had developed normally. Epilepsy and SUDEP, which occur by postnatal day 21 in Scn1b null animals, were observed within 20 days of induced Scn1b deletion in adult mice, suggesting that epileptogenesis in SCN1B‐DS does not result from defective brain development. Thus, the developmental brain defects observed previously in Scn1b null mice may model other co‐morbidities of DS.


Introduction
Voltage gated sodium channels (VGSCs) are heterotrimers, composed of one pore-forming a subunit and two nonpore-forming b subunits. 1 b1 subunits are ubiquitously expressed throughout the nervous system with wide subcellular localization in neurons where they associate with many, if not all, VGSC a subunits. 2 b1 subunits regulate VGSC a subunit trafficking, expression, and gating, associate with and modulate some voltage-gated potassium channels, and play nonconducting roles in neuronal pathfinding and fasciculation as immunoglobulin superfamily cell adhesion molecules (Ig-CAMs). 2 Variants in VGSC genes are linked to the developmental and epileptic encephalopathies (DEEs). 3 Dravet syndrome (DS) is a severe DEE predominantly linked to haploinsufficiency of SCN1A, encoding the VGSC a subunit Na v 1.1. Results from mouse models suggest that epilepsy in DS initiates from disinhibition through reduced sodium current density in cortical and hippocampal parvalbumin-positive GABAergic interneurons. 4 While the majority of DS patients have de novo, heterozygous variants in SCN1A, inherited, homozygous loss-of-function variants in SCN1B, encoding the VGSC b1/b1B subunits, also result in DS, or a DS-like DEE. 5,6 Scn1b null mice are a DS model, with severe seizures, ataxia, and 100% lethality prior to weaning. 5 Scn1b null mice have neuronal pathfinding defects in brain that precede the onset of seizures. 7 Based on these results, we proposed that epilepsy in this model is of neurodevelopmental origin due to the loss of b1-mediated Ig-CAM function. 7 In the related Scn1b-p.C121W homozygous mouse model, which has a ª 2019 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. similar DS-like phenotype, neuronal hyperexcitability was postulated to arise from the developmental loss of b1-mediated Ig-CAM function. 8 In support of this hypothesis, variants in other neurodevelopmental CAMs, for example, protocadherin 7 and 19, 3,8 are also associated with epilepsy.
Because b1 subunits are multifunctional Ig-CAMs and channel modulators, it is important to understand what roles altered development play in epileptogenesis in response to Scn1b loss-of-function. Here, we test the hypothesis that altered neuronal development is necessary for epileptogenesis in the Scn1b model of DS. We show that severe seizures and SUDEP occur following Scn1b deletion in adult mice that had developed normally, suggesting that epileptogenesis in SCN1B-DS does not result from defective brain development.

Animals
Experiments were performed in accordance with NIH guidelines and approved by the University of Michigan Institutional Animal Care and Use Committee. Scn1b Fl/Fl mice, on the C57BL/6J background, 9 were crossed with Slick-H mice (JAX Tg(Thy1-cre/ERT2,-EYFP)HGfng/ PyngJ, stock #012708) received on the (C57BL/6J x CBA) F1 background and backcrossed to C57BL/6J, to generate Slick-H/Scn1b Fl/Fl and Slick-H/Scn1b E/E mice. Tamoxifen (TMX, Sigma-Aldrich) was dissolved in sunflower oil + 2% ethanol at a final concentration of 10 lg/lL. 10 lL of TMX solution or vehicle per g mouse weight were administered intraperitoneal (IP) once per day for 4 consecutive days.
Immunohistochemistry P60 Slick-H mice, treated with TMX 10 days prior, were anesthetized with isoflurane and transcardially perfused with~10 mL of PBS followed by~10 mL of 4% paraformaldehyde (PFA). Tissues were postfixed overnight in 4% PFA, then cryoprotected in 10% sucrose followed by 30% sucrose overnight, flash frozen in 2-methylbutane, and stored at À80°C. 20 lm coronal brain sections were cut on a Leica cryostat and stored at À20°C until processing for immunohistochemistry.
For immunohistochemistry, sections at the same locations relative to Bregma for each animal were dried and postfixed for 10 min with 4% PFA, washed three times for 5 min each with 0.05 mol/L phosphate buffer (PB), and incubated in PBTGS blocking buffer (10% goat serum, 0.3% Triton X-100, 0.1 mol/L PB) for ≥2 h in a humidified chamber. Sections were then incubated with primary antibodies (diluted in PBTGS) overnight in a humidified chamber and washed 3 times for 10 min with 0.1 mol/L PB. Primary antibodies were anti-GABA (rabbit, Sigma-Aldrich, 1:400), anti-GFP (chicken, Invitrogen, 1:400), and anti-CTIP2 to identify cortical layer V/VI neurons (rat, Abcam, 1:250). From this point forward, all steps were performed in the dark to minimize photobleaching of secondary antibodies. Sections were incubated with goat anti-chicken AlexaFluor-488, goat anti-rat AlexaFluor-647, and/or goat anti-rabbit AlexaFluor-594conjugated secondary antibodies (Invitrogen, diluted in PBTGS) for 2 h, washed three times for 10 min in 0.1% PB, incubated with DAPI for 20 min, dried, and then coverslips were mounted using ProLong Gold anti-fade reagent (Invitrogen).
Sections were imaged using a Nikon A1R confocal microscope with Nikon NIS-Elements AR software located in the UM Department of Pharmacology using a 20 9 0.75 NA dry objective. Confocal images spanning 10 lm were acquired at 0.9 lm intervals and flattened using maximum signal for analysis. Images were processed and analyzed using NIH-ImageJ, and figures were assembled using Adobe Photoshop.

EEG Implantation
Slick-H/Scn1b Fl/Fl , Slick-H negative/Scn1b Fl/Fl , and Slick-H/ Scn1b E/E mice were implanted at P42 to 70 with 6 EEG electrodes. For surgery, each mouse was anesthetized with isoflurane or ketamine/xylazine and placed in a stereotaxic adapter. The electrodes were implanted over the left and right front lobes, the left and right parietal lobes, the cerebellum, and the sinus cavity using mounting screws. The electrodes were connected to a 6-pin electrode pedestal and the headcap was secured using dental cement. After 7 days of recovery, simultaneous EEG recording and video monitoring were performed on Ceegraph Vision; Bio-logic System Corporation and Natus recording systems. Following baseline recording for 2 days, mice were removed from recording for injection with TMX and recording was resumed at 7 days postinitial TMX injection. Signals were acquired at 256 Hz with simultaneous video monitoring. Data were filtered with a 1 Hz low pass filter, a 70 Hz high pass filter, and a notch filter at 60 Hz. Seizures were assessed manually.

Results
Scn1b null DS mice exhibit severe seizures beginning at postnatal day (P)10 and 100% lethality prior to~P21. 10 Our previous work suggested that aberrant brain development due to loss of b1-mediated cell adhesion may underlie epileptogenesis in this model. 7 To test this  1124 pyramidal neurons of layers 3, 5, and 6 ( Fig. 1A), as shown previously. 11 We co-stained with anti-GABA to examine whether GABA+ neurons are also targeted by Thy1 promoter-driven Cre recombinase in this strain. Neurons that were YFP+ were seldom GABA+ and vice versa (Fig. 1A, B, and F). We found similar results within the hippocampus, with dense labeling of the dentate gyrus and pyramidal neurons throughout the CA1, CA2, and CA3 regions, as well as in the subiculum, with limited overlap of YFP+ and GABA+ neurons ( Fig. 1C and D).
To investigate the developmental dependence of epilepsy and SUDEP in response to Scn1b deletion, we administered TMX to Slick-H/Scn1b Fl/Fl mice over various time ranges (P14-19, P42-117, and P > 100) to ask whether induced Scn1b deletion in adult mice that had developed normally resulted in seizures and death. Regardless of age of administration, TMX treatment resulted in severe epilepsy and death in 100% of Slick-H/ Scn1b Fl/Fl mice compared to controls (Slick-H negative Scn1b Fl/Fl mice injected with vehicle or Slick-H/Scn1b E/E mice injected with TMX) within 20 days ( Fig. 2A). All TMX-treated Slick-H/Scn1b Fl/Fl mice exhibited frequent spontaneous, behavioral seizures. To verify epileptic activity electrographically, we implanted three Slick-H/Scn1b Fl/ Fl mice, two Slick-H/Scn1b Fl/Fl vehicle control mice and two Slick-H negative Scn1b Fl/Fl control mice with EEG electrodes. Two days of baseline recording were followed by one IP administration of TMX or vehicle per day for four consecutive days to delete Scn1b. EEG recording was resumed 7 days after the initial TMX treatment (post-TMX). We observed no seizures in Slick-H/Scn1b Fl/Fl or control mice for the duration of baseline recording. In the Slick-H/Scn1b Fl/Fl group, seizures were detected as early as 7 days post-TMX ( Fig. 2B and C). Two mice exhibited severe epilepsy and were euthanized for ethical reasons at day 8 and 9, respectively. The third mouse had a more gradual increase in seizure severity and was euthanized at 20 days post-TMX due to morbidity. These results demonstrate that, although developmental brain defects are present in Scn1b-DS mice, epileptogenesis and SUDEP are not the result of impaired brain development.

Discussion
The results of this study demonstrate that epileptogenesis resulting from Scn1b loss-of-function is not due to impaired neuronal development. Furthermore, our data show that deletion of Scn1b in forebrain excitatory, but not inhibitory, neurons recapitulates the DS phenotype in mice. In previous work, targeted deletion of Scn1a in mouse inhibitory neurons phenocopied DS. 12 In contrast, targeted deletion of Scn1a in excitatory neurons failed to recapitulate the DS phenotype and instead ameliorated disease severity when coupled with deletion in interneurons. 13 Importantly, our work demonstrates that diverse mechanisms can lead to common neurological disease phenotypes.