Sodium channel SCN3A (Na_V1.3) regulation of human cerebral cortical folding and oral motor development

Human subjects and samples

Research performed on samples of human origin was conducted according to protocols approved by the institutional review boards of Boston Children’s Hospital, Beth Israel Deaconess Medical Center, Montreal Neurological Hospital and Institute, Erasmus MC UMC Rotterdam, and by The Children’s Hospital of University of Helsinki. Subjects were identified and evaluated in a clinical setting, and biological samples collected after obtaining written informed consent. Fetal brain tissue was received after release from clinical pathology, with a maximum post-mortem interval of 4 h. Cases with known anomalies were excluded. Male and female brain tissue was collected from 17–20 gestation weeks. Tissue was transported in HBSS medium on ice to the laboratory for research processing.

Phenotypic assessment

All affected individuals and clinical data were examined by neurologists and radiologists, and polymicrogyria was diagnosed using criteria described previously (Guerrini et al., 2000). Detailed clinical information on affected individuals in Family A was collected over a 20-year period, summarized in Table S1. The affected individuals of Families B - F were evaluated by neurologists over many years and detailed clinical and radiographic evaluations are described in supplemental results text.

Clinical description of families

Family A, a multigenerational Finnish family, is the largest family with bilateral perisylvian polymicrogyria (PMG) reported to date (Guerreiro et al., 2000), having four pairs of affected siblings with variable penetrance and expression of PMG and oral motor dysfunction (dysarthric speech and deficient tongue movement) (Figures 1B & C; Table S1). With the exception of a single rolandic seizure in one individual (A12), neurological evaluation of the affected individuals performed throughout life confirmed the absence of epilepsy in Family A. Two affected individuals (A11 and A12) showed slightly subnormal language comprehension and intellectual ability, and these features ranged from borderline normal to subnormal in others (Figure 1C; Table S1).

Family B is a multigenerational French Canadian family with bilateral perisylvian PMG and oral motor dyspraxia (Figures 1B & C). As previously described, the two affected siblings (B02 and B03) had delayed speech development and a history of choking in infancy, and have dysarthria, drooling and significant restriction of tongue movements. Additionally, they were both noted to have difficulty whistling and blowing, abnormal brisk jaw jerk, increased reflexes, absent gag reflex and automatic involuntary dissociation of facial movements (Guerreiro et al., 2000). Upon reevaluation at 43 years of age, B03 had macrocephaly with a head circumference of 57 cm (99^th percentile, 2.4 standard deviations), her height was 163 cm (48^th percentile) and her weight was 126 pounds (49^th percentile). She reported a history of having difficulty manipulating objects in childhood, and was observed to have normal muscle tone and gross motor movements, but had a mild tremor at the end of intentional movements and mild difficulty with rapid alternating movements. Prior clinical testing for B03 included a normal karyotype (46, XX) and normal IQ. B02 was evaluated on the same occasion, was 50 years of age, and presented with a very similar clinical picture as his sister, B03. B01, the daughter of B02 and previously described to have mild dysarthria and a normal brain MRI (Guerreiro et al., 2000), was reported at 16 years of age to have intellectual disability but no speech disorder. A sister of B02 and B03 died at 16 months of age of unknown cause, and a maternal uncle never developed speech, had epilepsy and died in adulthood. Brain imaging was not available for either the deceased sister or uncle. B04, sister of B02 and B03, was unaffected. B05, the mother of B02 and B03, was unaffected and had a normal brain MRI (Guerrini et al., 2000) and normal fluorescence in situ hybridization study of the DiGeorge syndrome region [ish 22q11.2 (TUPLE1×2)].

Family C is of Dutch descent and has a single male affected with unilateral perisylvian PMG and contralateral abnormal perisylvian sulcation with developmental motor and speech delay (Figure 1B & C). The proband has two healthy older sisters and healthy parents. He was born by induced delivery at 41 weeks gestation, following an uncomplicated pregnancy, and had a birth weight of 3.5 kg (46^th percentile). In infancy, he was unable to breastfeed and learning to chew was slow and required therapy. He presented at 1 year of age with delay of motor development and hypotonia. A brain MRI was obtained at 2 years of age, noting right-sided perisylvian PMG, involving the posterior-frontal, anterior-parietal and postero-superior temporal lobes. The posterior body and isthmus of the corpus callosum was thin, the right cerebral ventricle was mildly enlarged and white matter volume was slightly diminished in the right hemisphere. At 3 years of age, his height is 93 cm (-1.5 standard deviations) and head circumference is 52.0 cm (0.8 standard deviations). He is noted to have a prominent forehead and downward-slanting eyelids. Neurologic examination revealed axial hypotonia and very mild left pyramidal signs but no differences in strength. Vision and hearing are normal; he sleeps well and has not had any seizures. At 3 years, he is able to stably walk and tries to jump and run, has good social skills but weak executive functions. Speech is delayed, as he uses two to three word sentences only, but oral motor abilities, including articulation, swallowing and chewing, are normal. He has a normal gag reflex and no mouth breathing.

Family D is of mixed European descent (Poland, Germany and Ireland) and has a single male affected with frontoparietal PMG, seizures, microcephaly, and neurodevelopmental delay (Figures 1B & C). The proband was born at 42 weeks gestation following an uncomplicated pregnancy and presented with seizures at 2 weeks of age. At 2 months of age, he had a normal head circumference and length (40^th and 80^th percentiles respectively by report), a small penis (<10^th percentile reportedly), was non-dysmorphic, exhibited hypotonia, had feeding difficulties and apneic episodes, as well as developmental delay (not smiling or rolling). Brain MRI at 2 months of age revealed extensive coarse PMG in the frontal and parietal lobes with subcortical calcifications and abnormal frontal white matter. A thin but fully formed corpus callosum was noted and the white matter was markedly diminished, especially frontally. Upon evaluation at 1 year, he had a mixed seizure disorder with complex and simple partial seizures described as occurring 1 per day initially, worsening when he was ill, and better controlled more recently. There was significant generalized hypotonia with spasticity and upper extremity hypertonia, and he did not have complete head control. He had oral motor discoordination (unable to bottle feed but able to breastfeed), recurrent otitis media and genu valgum that did not require intervention. Developmentally, he was rolling over at 1 year but did not fully grab, hold or transfer toys from his left to right hand; he said “ma” and responded to his name but did not fix or follow consistently. At that time he received physical, occupational and vision therapies. Head circumference at 10 years 10 months was 49 cm (3.89 standard deviations below the mean). Ophthalmologic evaluation noted strabismus and significant cortical visual impairment with normal retina and optic nerves. At 11 years 8 months of age he has remained non-verbal, with dysphagia and mixed seizure disorder, hypotonia and severe global delay.

Family E is a non-consanguineous family from the United Arab Emirates and has a single female affected with bilateral frontoparietal PMG, microcephaly and severe global developmental delay (Figures 1B & C). The proband was born at full term following an unremarkable pregnancy and delivery, and her birth weight was 3 kg (21^st percentile; -0.8 standard deviations). Hypotonia and developmental delay prompted a brain MRI at 5 months of age, which revealed bilateral PMG sparing the posterior temporal and occipital lobes; the cerebral ventricles were mildly enlarged and white matter volume slightly reduced. At 10 months of age, she had microcephaly; her head circumference was 40.7 cm (-3.2 standard deviations). At that time she was able to roll over but not sit or crawl, she had a palmar grasp only and had stereotyped movements. Neurologic evaluation revealed right esotropia, no response to visual or auditory stimuli, generalized hypotonia with head lag and normal deep tendon reflexes. At 13 months, she had not made further motor developmental progress but responded well to visual and auditory stimuli, and was social. She did not have seizures, though an EEG was reported to have shown bilateral synchronous epileptiform discharges but no medication was prescribed. Karyotype and serum lactic acid and ammonia levels were reported as normal. Another brain MRI was performed at 9 years of age that reported a thickened cortex bilaterally, shallow Sylvian fissures, slightly prominent cerebral ventricles, sparse white matter of normal signal intensity and minimally increased extra-axial cerebral spinal fluid. Follow-up evaluation at 13 years of age revealed spastic quadriplegia and global developmental delay. She was wheelchair bound, could roll over but not sit up or stand. She was nonverbal, had excessive drooling and feeding difficulties, taking soft and pureed foods orally with slow swallowing. Decreased range of motion, flexion deformity of the left wrist, equinus deformity of the left foot, and significant scoliosis with right curvature were noted. She was observed to be very attentive and smiling, and to both fix and follow appropriately. She was reported to menstruate, have urinary incontinence and have sleep disturbances.

Family F is of French Canadian, Russian and Irish descent and has a single female affected with unilateral right-sided PMG and seizures (Figures 1B & C). The proband has three siblings and a father who are healthy, and her mother has a history of thyroid adenoma and left frontal meningioma. She was born at term, had a birth weight of 4.9 kg (>99^th percentile, 3.2 standard deviations), and had an uncomplicated neonatal course. Seizures were first noted at 2 years of age and she had a history of motor developmental delay but normal speech and oral motor development. Her ability to reason and to comprehend others’ language was reportedly reduced.

Brain MRI at 21 years of age revealed right hemispheric PMG with the right hemisphere being smaller than the left, fully formed and thick corpus callosum, small right basal ganglia and thalamus, significant enlargement of the right lateral ventricle and severe diminution of the right hemispheric white matter. On evaluation at 28 years of age, she had macrocephaly with a head circumference of 60.5 cm (5.1 standard deviations), her height was 168 cm (77^th percentile, 0.74 standard deviations), and her weight was 188 lbs (95^th percentile, 1.7 standard deviations). Notably, her mother was also macrocephalic with a head circumference of 61 cm (6 standard deviations). Treated with Tegretol, a seizure frequency of once or twice a year was reported. She had good vision with normal visual field and extraocular movements.

Human genetics

Leukocyte-derived DNA was utilized for studies of all families. Genome-wide genotyping was performed on individuals 1–12 in Family A using the Illumina Infinium HumanHap550-Duo BeadChip. These data were used to perform linkage and haplotype analysis. Genotypes were called using GenomeStudio Software (Illumina). Low performing SNPs (those not called in all individuals in this study or not called in >5% of individuals run on the same chips), low variance SNPs (those with minor allele frequencies (MAF) of 0 in all individuals in this study; or that have an MAF < 10% or that deviate from HWE with P-value < 0.00000001 among unrelated individuals run on the same chips), and SNPs with Mendelian and non-Mendelian errors (calculated with Merlin) were removed from the analysis using PLINK. PLINK was used to thin SNPs to reduce linkage disequilibrium. Linkage analysis was performed to generate a LOD score from the remaining SNPs using Merlin under a dominant mode of inheritance assuming a disease allele prevalence of 0.0001 and a penetrance of 0.6. Haplotype analysis was performed on the SNPs around the maximum LOD score using Merlin. Whole exome sequencing (WES) was performed at the Broad Institute using Agilent Sure-Select Human All Exon v2.0 capture array and sequenced on Illumina HiSeq2000 sequencers.

Identification and segregation of alleles

Linkage and haplotype analysis in Family A identified a candidate locus at Chromosome 2 (Figures S1B & C) with the highest LOD score of 2.48 at rs10930488-rs1489635; chr2:164,028,745–172,490,226 (hg19). Targeted sequencing was performed and the mutation identified in Family A is Chr2(GRCh37):g.165947387A>T, NM_006922.3:c.5276T>A, p.Phe1759Tyr (Figure S1A). Algorithms suggest that the mutation is of a highly conserved nucleotide (phyloP: 5.29 [-14.1;6.4]), and predict the amino acid change to be deleterious [SIFT: deleterious (score: 0, median: 4.32), MutationTaster: disease-causing (p-value: 1), PolyPhen-2: probably damaging (score: 1.0)]. The mutation identified in Family B is Chr2(GRCh37):g.165953971T>G, NM_006922.3:c.4030A>C, p.Ile1344Leu. Algorithms suggest that the mutation is of a highly conserved nucleotide (phyloP: 5.21 [-14.1;6.4]), and predict the amino acid change to be deleterious [SIFT: deleterious (score: 0, median: 4.32), MutationTaster: disease-causing (p-value: 1), PolyPhen-2: possibly damaging (score: 0.88)].

The de novo mutation identified in Family C is Chr2(GRCh37):g.165987770A>G, NM_006922.3:c.2549T>C, p.Leu850Pro. Algorithms suggest that the mutation is of a highly conserved nucleotide (phyloP: 5.13 [-14.1;6.4]), and predict the amino acid change to be deleterious [SIFT: Deleterious (score: 0, median: 4.32), MutationTaster: disease-causing (pvalue: 1), PolyPhen-2: probably damaging (score: 0.999). The de novo mutation in Families D and E is Chr2(GRCh37):g165986748A>G, NM_006922.3:c.2624T>C, p.Ile875Thr. Algorithms suggest that the mutation is of a highly conserved nucleotide (phyloP: 4.89 [-14.1;6.4]), and predict the amino acid change to be deleterious [SIFT: Deleterious (score: 0.01, median: 4.32), MutationTaster: disease causing (p-value: 1), PolyPhen2: probably damaging (score: 1.0)]. I875 was previously reported in two large intellectual and developmental disability cohorts, ClinVar ID 373960 (Deciphering Developmental Disorders Study, 2017; Lelieveld et al., 2016). The mutation in Family F is Chr2(GRCh37):g.165997318C>T, NM_006922.3:c.1862G>A, p.Arg621His. Algorithms suggest that the mutation is of a highly conserved nucleotide (phyloP: 4.48 [-14.1;6.4]), and predict the amino acid change to be deleterious [SIFT: Deleterious (score: 0, median: 4.32), MutationTaster: disease causing (prob: 1), PolyPhen2: probably damaging (score: 0.99)].

Sanger sequencing confirmed familial segregation of the inherited mutations in Family A and B, and the de novo occurrence of the mutations in Family C, D, and E, being absent from both parents (Figures S1F & H). In Family F, the mutation was confirmed by Sanger sequencing in the proband and was absent from her mother but DNA was not available from the father to verify its de novo occurrence (Figure S1G). Further, amino acids upstream and downstream of R621, L850, I875, I1344 and F1759 in SCN3A are highly conserved in orthologs as well, 16 of 20 amino acids, 19 of 20 amino acids, 19 of 20 amino acids, 20 of 20 amino acids, and 20 of 20 amino acids respectively (Figures S1 D-H). Taken together, these data demonstrate that the SCN3A gene and the families’ mutation sites and surrounding alleles are highly conserved.

Electrophysiology

Neurons were visualized using an inverted Olympus IX73 microscope with epifluorescence capabilities. Whole-cell voltage-clamp currents (series resistance compensated 85%) were recorded with an Axopatch 200B amplifier and pClamp 9.2 (Molecular Devices, Sunnyvale, CA). Glass pipettes were formed from borosilicate glass (1.5–3 Mohm) on a Sutter Puller (P-97 Sutter Instrument) and filled with intracellular solution for voltage clamp (in mM: 80 CsMES, 25 NaCl, 10 HEPES, 10 Cs4-BAPTA, 2 MgCl2, free Ca 90uM, OsM 295 mOs. pH 7.3 with CsOH) or for current clamp (in mM: 120 K-gluconate, 10 Na-gluconate, 4 NaCl, 10 HEPES-K, 10 Na phosphocreatine, 2 Na-ATP, 4 Mg-ATP, and 0.3 GTP, adjusted to pH 7.3 with KOH). Control extracellular solution contained the following (in mM): 150 NaCl, 10 HEPES, 1.8 CaCl2, pH to 7.4 with NaOH, 300mOsM.

HEK293T cells (from ATCC, CRL-3216) were grown under standard culture conditions (5% CO2, 37°C) in Dulbeccos's Modified Eagle's Medium with 10% FBS and antibiotics. The sex of the cell line was undetermined. Site directed mutagenesis of SCN3A-F1759Y and SCN3A-I875T was performed using a bridge PCR method followed by Sanger sequencing of the entire plasmid to control for off-target mutations. Cells were transfected (Lipofectamine 3000, ThermoFisher) with one of the SCN3A alpha subunits: SCN3A-WT, SCN3A-F1759Y, or SCN3A-I875T, together with the human auxiliary Na_V-Beta subunits (β1-IRES-CD4 and β2-IRES-GFP; using a plasmid ratio of 1:6 for β:SCN3A) to support membrane localization and physiological current kinetics. Data were analyzed with IGOR-Pro (Wavemetrics) and reported as mean ± S.E.M. Conductancevoltage plots (from measured reversal potentials) were fit with a Boltzmann function of the following form (IImax/{1exp[(VVhalf)/ks]}), where Imax is the maximal current, V is the prepulse potential, Vhalf is the half activation potential of the current, and ks is the slope factor.

Statistical significance was assessed with Student’s two-tailed t-test. For measuring the noninactivating persistent current (I-Na_P), the mean current from the last 30 ms of the voltage step was measured and this value was divided by the maximal peak inward current. These I-Na_P data are presented as a percentage of the maximal peak inward current.

Human Tissue Brain Preparation and mRNA in situ hybridization

Following fixation (4% PFA) and cryoprotection (30% sucrose), brains (17 – 20 gestational weeks, both male and female) were frozen using Isopentane on dry ice. Samples were sectioned at 20 – 30 μm thickness (Leica Cryostat), mounted immediately onto warm charged SuperFrost Plus slides (Fisher), then dried at -20°C for 20 minutes before storage at –80°C. After applying a hydrophobic barrier around the tissue (ImmEdge Pen, Vector Labs), sections were baked for 20 minutes at 60°C before beginning with the manufacturer’s standard protocol for multiplex fluorescent in situ hybridization (Multiplex Version 2 kit, Advanced Cell Diagnostics). In situ probe ACD catalog numbers are as follows: Vimentin: 479411, SCN3A: 460121, GFAP: 311801, RBFOX: 415591, Eomes: 429691. Whole tissue mRNA in situ imaging was performed on a Zeiss Axio Observer with image tiling at 20X magnification. Bright-field images were background corrected by Zen Blue Software for center intensity illumination and stitched together (Figure 3C, D).

Ferret in utero electroporation

All animal experimentation was carried out under protocols approved by the IACUC of Boston Children’s Hospital. Ferrets (Mustela putorius furo) were obtained from Marshall Bioresources and animals were same-sex housed within a dedicated Large Animal Facility of Boston Childrens Hospital on a 12-hr light/dark cycle at 18°C–23°C, with rotating sensory enrichment. Food and water were available ad libitum. In utero electroporations were performed according to standard procedures (Kawasaki et al., 2012). Briefly, pregnant Jills (embryonic day 32–33) were anesthetized with 5% isoflurane and maintained at 3% utilizing a nose cone. Following laparotomy, 1.5–3μl (2 μg/μl) of the SCN3A and mCherry constructs were introduced into the lateral ventricle using a pulled glass micropipette and Hamilton syringe (Hamilton Company). 150V square electric pulses were passed 5 times at 1s intervals (ECM830, BTX). Tissues were harvested on postnatal days P0 and ~P33, and fixed in 4% PFA. Successful plasmid expression in embryos was evaluated based on mCherry fluorescence (Figure S4B). Both male and female embryos were targeted for injection.

Ferret Tissue Preparation and Immunohistochemistry

Following perfusion with ice cold 4% PFA in PBS, tissues were drop-fixed for 8 hours in 4% PFA at 4°C. For cryosections, brains were cryoprotected in 30% sucrose overnight, embedded in OCT, frozen in isopentane, sectioned at 15 – 35 um thickness (Leica Cryostat), and mounted onto charged SuperFrost Plus slides (Fisher). After applying a hydrophobic barrier around the tissue (ImmEdge Pen, Vector Labs), slides were washed in cold 0.1M PB followed by antigen retrieval and endogenous fluorescence quenching (Retrievagen A pH6.0, BD Biosciences) at 80–90°C in a hybridization oven for 45 minutes. Sections were then cooled to room temperature in Retrievagen A, washed in cold 0.1M PB, and blocked for 1 hour at room temperature (5% Normal Donkey Serum, 1% w/v BSA, 0.2% w/v each glycine/lysine, in PBS). Slides were incubated with primary antibodies for two nights on a rotary shaker at 4°C in blocking buffer plus 0.3% Triton X-100, washed in PBS, and incubated for two hours at room temperature with secondary antibodies diluted in blocking buffer (1:500; Jackson Immunoresearch). Finally, slides were washed in PBS, and coverslipped with Fluoromount-G containing Dapi (Southern Biotech). Images were obtained with a Zeiss LSM700 confocal microscope and Leica MZ16 F fluorescence stereomicroscope. Primary antibodies include mouse anti-phosphorylated Vimentin (4A4; 1:500; MBL), rabbit anti-Ki67 (1:250; Abcam ab15580), chicken anti-GFAP (1:1000; Abcam ab4674), mouse anti-TUJ1 (beta-III tubulin; 1:500; Abcam ab7751), rabbit anti-RFP (1:250; Rockland 600–901-379S), rabbit anti-SATB2 (1:400; Bethyl A301–864A). Secondary antibodies included AlexaFluor (1:500; 488nm, 563nm, 650nm) donkey anti-mouse and donkey anti-rabbit (Invitrogen).

Membrane protein extraction and immunoblotting

HEK293T cells were harvested and membranes were isolated according to manufacturer’s instructions (Mem-PER Plus Membrane Protein Extraction Kit for Mammalian Cells, Thermo Fisher Scientific). Membrane proteins (25ug) were resolved on 4–15% SDS-polyacrylamide gels (Bio-Rad), transferred to PVDF membrane (GE Healthcare Life Science), and probed with rabbit anti-Pan Na Channel α Subunit (clone D219C, 1:1000, Cell Signaling Technology), mouse antiATP1A1 (clone 464.6, 1:1000, Thermo Fisher Scientific), and chicken anti-GFP (1:1000, Abcam) antibodies, followed by anti-rabbit or anti-mouse horseradish peroxidase secondary antibodies (1:2000, Cell Signaling Technology). Immunoblots were developed with the Amersham ECL Western Blotting Detection Kit (GE Healthcare Life Science).

Quantitative MRI analysis

The MRI images (B03 and control age-matched subject) were processed to extract the surfaces of the gray/white matter and gray matter/cerebrospinal fluid boundaries, and to define Brodmann areas on the cortical surface using FreeSurfer software.

Human Gene Expression Analysis

The Allen Human Brain Atlas (ABA) publishes a rich dataset of cortical genetic expression across cortical brain regions, from age 8 weeks post conception to adult ages (Jones et al., 2009). BrainSpan data analysis of SCN3A (ch2:165944030–166060577, GRCh37/hg19) and SCN1A (ch2: 166,845,670–166,930,180, GRCh37/hg19) was performed. RNA-seq expression measured in RPKM (reads per kilobase exon per million mapped reads) was obtained from the BrainSpan project data and summarized to Gencode v10 exons for all annotated neocortical tissues aged 8 weeks post conception to 38 years. Cortical areas for Figure 3A include: dorsolateral prefrontal cortex; ventrolateral prefrontal cortex; anterior (rostral) cingulate (medial prefrontal) cortex; orbital frontal cortex; primary motor-sensory cortex; parietal neocortex; posterior (caudal) superior temporal cortex (area 22c), inferolateral temporal cortex (area 20); occipital neocortex.

Analysis of Single Cell RNA-seq Data

Dataset was provided by Alex Pollen UCSF; see (Pollen et al., 2015) methods for additional information. Briefly, fetal cortex was dissociated and captured on C1 (Fluidigm). Classification of cells was completed using principal component analysis (PCA) and used expectation-maximization clustering to group cells based on their position in PC space. Gene expression values were normalized based on library size as counts per million reads (CPM) and log₂(CPM+1) transformed. Libraries with fewer than 1,000 genes detected above 1 CPM were eliminated as outliers. Since these data contain many cells with zero CPM for the genes of interest, either due to lack of expression or to the limits of detection of single cell RNA-seq, a two-part Wilcoxon test was used to test differences in gene expression. A compound null hypothesis of no difference in the proportion of cells with zero CPM and of no difference in the mean CPM of the non-zero CPM cells for a given gene between two groups was tested. An R code, which applies a two-part Wilcoxon test with a continuity-corrected binomial test was implemented. These tests were conducted with a Bonferroni adjusted alpha level of 0.017 (0.05/3). Data are presented as box-plots.

Primary Neuronal Cultures and Neurite Analysis

Neuronal cultures were prepared according to established protocols with modifications. Briefly, the perisylvian cortex was dissected from 20 WKGS human samples, male and female cases, and dissociated according to the manufacturer’s protocol into a single cell suspension (MACs neural dissociation kit, Miltenyi Biotec). Cortical neurons were plated onto poly-ornithine and laminin treated coverslips at 5 × 10⁴ cells/well in 24-well plates and maintained in DMEM/Neurobasal, N2, B27, glucose, NaHCO3, HEPES (4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid) with fibroblast growth factor 2 (FGF-2; 20 ng/ml; Invitrogen) and EGF (FGF-2; 20 ng/ml; Invitrogen) treatment for the first 24 h only. Neurons were transfected with mCherry vector together with each experimental condition at a 1:6 plasmid ratio as described above (Figure S2). Cultured medium was changed every other day. Neurons were fixed after 5 days in vitro (DIV 5) with 4% PFA in PBS for 15 min. at room temperature. Cells were blocked in 10% NGS in PBS with 0.3% Triton X-100 for 1 h at room temperature followed by immunostaining. Images were captured using a digital camera under a Zeiss Axio Observer fluorescent microscope. 30–50 neurons were analyzed from three separate coverslips for each experimental condition. Neurites that had lengths at least twice the diameter of the cell body were measured. Neurite lengths from the soma were traced and measured using ImageJ software in a blinded manner, and data were analyzed using Igor Pro Software. Statistical significance was assessed with Student’s two-tailed t-test.

Ferret Brain Magnetic Resonance Imaging

Three P30 ferret brains (expressing SCN3A-WT, SCN3A-F1759Y, and mCherry) were post-fixed in 4% paraformaldehyde in PBS and submerged into perfluorocarbon oil (Fomblin, Fisher Scientific) at 4°C for 3 days. The brains were scanned using a Bruker BioSpec 70/30 7T MRI scanner (Small Animal Imaging Laboratory) at BCH. This is a high-resolution (sub-millimeter) MRI scanner designed specifically for imaging small animals, and has a 30 cm bore and 450 mT/m gradient. Ferret MRI scans are isotropic 63μm voxels across the entire brain. Cortical gray matter and white matter were visualized using the FreeSurfer and OsiriX software.