Stuttering associated with a pathogenic variant in the chaperone protein cyclophilin 40.

Stuttering is a common speech disorder that interrupts speech fluency and tends to cluster in families. Typically, stuttering is characterized by speech sounds, words or syllables which may be repeated or prolonged and speech that may be further interrupted by hesitations or 'blocks'. Rare variants in a small number of genes encoding lysosomal pathway proteins have been linked to stuttering. We studied a large four-generation family in which persistent stuttering was inherited in an autosomal dominant manner with disruption of the cortico-basal-ganglia-thalamo-cortical network found on imaging. Exome sequencing of three affected family members revealed the PPID c.808C>T (p.Pro270Ser) variant that segregated with stuttering in the family. We generated a Ppid p.Pro270Ser knock-in mouse model and performed ex vivo imaging to assess for brain changes. Diffusion-weighted MRI in the mouse revealed significant microstructural changes in the left corticospinal tract, as previously implicated in stuttering. Quantitative susceptibility mapping also detected changes in cortico-striatal-thalamo-cortical loop tissue composition, consistent with findings in affected family members. This is the first report to implicate a chaperone protein in the pathogenesis of stuttering. The humanized Ppid murine model recapitulates network findings observed in affected family members.

Selective NaV1.1 activation rescues Dravet syndrome mice from seizures and premature death.

Dravet syndrome is a catastrophic, pharmacoresistant epileptic encephalopathy. Disease onset occurs in the first year of life, followed by developmental delay with cognitive and behavioral dysfunction and substantially elevated risk of premature death. The majority of affected individuals harbor a loss-of-function mutation in one allele of SCN1A, which encodes the voltage-gated sodium channel NaV1.1. Brain NaV1.1 is primarily localized to fast-spiking inhibitory interneurons; thus the mechanism of epileptogenesis in Dravet syndrome is hypothesized to be reduced inhibitory neurotransmission leading to brain hyperexcitability. We show that selective activation of NaV1.1 by venom peptide Hm1a restores the function of inhibitory interneurons from Dravet syndrome mice without affecting the firing of excitatory neurons. Intracerebroventricular infusion of Hm1a rescues Dravet syndrome mice from seizures and premature death. This precision medicine approach, which specifically targets the molecular deficit in Dravet syndrome, presents an opportunity for treatment of this intractable epilepsy.

In situ 3D visualization of biomineralization matrix proteins.

Calcium biominerals occur in all major animal phyla, and through biomolecular control, exhibit such diverse structures as exoskeletons, shells, bones, teeth and earstones (otoliths). Determining the three-dimensional expression of key biomineral proteins, however, has proven challenging as typical protein identification methods either lose spatial resolution during dissolution of the mineral phase or are costly and limited to two-dimensional expression of high abundance proteins. Here we present a modification of the CLARITY and ACT-PRESTO protocols to visualize and confirm, for the first time, the timing of expression and function of two key regulators of biomineralization.

'Neonatal' Nav1.2 reduces neuronal excitability and affects seizure susceptibility and behaviour.

Developmentally regulated alternative splicing produces 'neonatal' and 'adult' isoforms of four Na(+) channels in human brain, NaV1.1, NaV1.2, NaV1.3 and NaV1.6. Heterologously expressed 'neonatal' NaV1.2 channels are less excitable than 'adult' channels; however, functional importance of this difference is unknown. We hypothesized that the 'neonatal' NaV1.2 may reduce neuronal excitability and have a seizure-protective role during early brain development. To test this hypothesis, we generated NaV1.2(adult) mice expressing only the 'adult' NaV1.2, and compared the firing properties of pyramidal cortical neurons, as well as seizure susceptibility, between the NaV1.2(adult) and wild-type (WT) mice at postnatal day 3 (P3), when the 'neonatal' isoform represents 65% of the WT NaV1.2. We show significant increases in action potential firing in NaV1.2(adult) neurons and in seizure susceptibility of NaV1.2(adult) mice, supporting our hypothesis. At postnatal day 15 (P15), when 17% of the WT NaV1.2 is 'neonatal', the firing properties of NaV1.2(adult) and WT neurons converged. However, inhibitory postsynaptic currents in NaV1.2(adult) neurons were larger and the expression level of Scn2a mRNA was 24% lower compared with the WT. The enhanced seizure susceptibility of the NaV1.2(adult) mice persisted into adult age. The adult NaV1.2(adult) mice also exhibited greater risk-taking behaviour. Overall, our data reveal a significant impact of 'neonatal' NaV1.2 on neuronal excitability, seizure susceptibility and behaviour and may contribute to our understanding of NaV1.2 roles in health and diseases such as epilepsy and autism.

The antiepileptic medications carbamazepine and phenytoin inhibit native sodium currents in murine osteoblasts.

OBJECTIVE: Fracture risk is a serious comorbidity in epilepsy and may relate to the use of antiepileptic drugs (AEDs). Many AEDs inhibit ion channel function, and the expression of these channels in osteoblasts raises the question of whether altered bone signaling increases bone fragility. We aimed to confirm the expression of voltage-gated sodium (NaV ) channels in mouse osteoblasts, and to investigate the action of carbamazepine and phenytoin on NaV channels. METHODS: Immunocytochemistry was performed on primary calvarial osteoblasts extracted from neonatal C57BL/6J mice and additional RNA sequencing (RNASeq) was included to confirm expression of NaV . Whole-cell patch-clamp recordings were made to identify the native currents expressed and to assess the actions of carbamazepine (50 µm) or phenytoin (50 µm). RESULTS: NaV expression was demonstrated with immunocytochemistry, RNA sequencing, and functionally, with demonstration of robust tetrodotoxin-sensitive and voltage-activated inward currents. Application of carbamazepine or phenytoin resulted in significant inhibition of current amplitude for carbamazepine (31.6 ± 5.9%, n = 9; p < 0.001), and for phenytoin (35.5 ± 6.9%, n = 7; p < 0.001). SIGNIFICANCE: Mouse osteoblasts express NaV , and native NaV currents are blocked by carbamazepine and phenytoin, supporting our hypothesis that AEDs can directly influence osteoblast function and potentially affect bone strength.

Reduced dendritic arborization and hyperexcitability of pyramidal neurons in a Scn1b-based model of Dravet syndrome.

Epileptic encephalopathies, including Dravet syndrome, are severe treatment-resistant epilepsies with developmental regression. We examined a mouse model based on a human ß1 sodium channel subunit (Scn1b) mutation. Homozygous mutant mice shared phenotypic features and pharmaco-sensitivity with Dravet syndrome. Patch-clamp analysis showed that mutant subicular and layer 2/3 pyramidal neurons had increased action potential firing rates, presumably as a consequence of their increased input resistance. These changes were not seen in L5 or CA1 pyramidal neurons. This raised the concept of a regional seizure mechanism that was supported by data showing increased spontaneous synaptic activity in the subiculum but not CA1. Importantly, no changes in firing or synaptic properties of gamma-aminobutyric acidergic interneurons from mutant mice were observed, which is in contrast with Scn1a-based models of Dravet syndrome. Morphological analysis of subicular pyramidal neurons revealed reduced dendritic arborization. The antiepileptic drug retigabine, a K+ channel opener that reduces input resistance, dampened action potential firing and protected mutant mice from thermal seizures. These results suggest a novel mechanism of disease genesis in genetic epilepsy and demonstrate an effective mechanism-based treatment of the disease.

Visualization of mouse barrel cortex using ex-vivo track density imaging.

We describe the visualization of the barrel cortex of the primary somatosensory area (S1) of ex vivo adult mouse brain with short-tracks track density imaging (stTDI). stTDI produced much higher definition of barrel structures than conventional fractional anisotropy (FA), directionally-encoded color FA maps, spin-echo T1- and T2-weighted imaging and gradient echo T1/T2*-weighted imaging. 3D high angular resolution diffusion imaging (HARDI) data were acquired at 48 micron isotropic resolution for a (3mm)(3) block of cortex containing the barrel field and reconstructed using stTDI at 10 micron isotropic resolution. HARDI data were also acquired at 100 micron isotropic resolution to image the whole brain and reconstructed using stTDI at 20 micron isotropic resolution. The 10 micron resolution stTDI maps showed exceptionally clear delineation of barrel structures. Individual barrels could also be distinguished in the 20 micron stTDI maps but the septa separating the individual barrels appeared thicker compared to the 10 micron maps, indicating that the ability of stTDI to produce high quality structural delineation is dependent upon acquisition resolution. Close homology was observed between the barrel structure delineated using stTDI and reconstructed histological data from the same samples. stTDI also detects barrel deletions in the posterior medial barrel sub-field in mice with infraorbital nerve cuts. The results demonstrate that stTDI is a novel imaging technique that enables three-dimensional characterization of complex structures such as the barrels in S1 and provides an important complementary non-invasive imaging tool for studying synaptic connectivity, development and plasticity of the sensory system.

Mapping somatosensory connectivity in adult mice using diffusion MRI tractography and super-resolution track density imaging.

In this study we combined ultra-high field diffusion MRI fiber tracking and super-resolution track density imaging (TDI) to map the relay locations and connectivity of the somatosensory pathway in paraformaldehyde fixed, C57Bl/6J mouse brains. Super-resolution TDI was used to achieve 20 µm isotropic resolution to inform the 3D topography of the relay locations including thalamic barreloids and brainstem barrelettes, not described previously using MRI methodology. TDI-guided mapping results for thalamo-cortical connectivity were consistent with thalamo-cortical projections labeled using virus mediated fluorescent protein expression. Trigemino-thalamic TDI connectivity maps were concordant with results obtained using anterograde dye tracing from brainstem to thalamus. Importantly, TDI mapping overcame the constraint of tissue distortion observed in mechanically sectioned tissue, enabling 3D reconstruction and long-range connectivity data. In conclusion, our results showed that diffusion micro-imaging at ultra-high field MRI revealed the stereotypical pattern of somatosensory connectivity and is a valuable tool to complement histologic methods, achieving 3D spatial preservation of whole brain networks for characterization in mouse models of human disease.

Mono and biallelic variants in HCN2 cause severe neurodevelopmental disorders.

Hyperpolarization activated Cyclic Nucleotide (HCN) gated channels are crucial for various neurophysiological functions, including learning and sensory functions, and their dysfunction are responsible for brain disorders, such as epilepsy. To date, HCN2 variants have only been associated with mild epilepsy and recently, one monoallelic missense variant has been linked to developmental and epileptic encephalopathy. Here, we expand the phenotypic spectrum of HCN2- related disorders by describing twenty-one additional individuals from fifteen unrelated families carrying HCN2 variants. Seventeen individuals had developmental delay/intellectual disability (DD/ID), two had borderline DD/ID, and one had borderline DD. Ten individuals had epilepsy with DD/ID, with median age of onset of 10 months, and one had epilepsy with normal development. Molecular diagnosis identified thirteen different pathogenic HCN2 variants, including eleven missense variants affecting highly conserved amino acids, one frameshift variant, and one in-frame deletion. Seven variants were monoallelic of which five occurred de novo, one was not maternally inherited, one was inherited from a father with mild learning disabilities, and one was of unknown inheritance. The remaining six variants were biallelic, with four homozygous and two compound heterozygous variants. Functional studies using two-electrode voltage-clamp recordings in Xenopus laevis oocytes were performed on three monoallelic variants, p.(Arg324His), p.(Ala363Val), and p.(Met374Leu), and three biallelic variants, p.(Leu377His), p.(Pro493Leu) and p.(Gly587Asp). The p.(Arg324His) variant induced a strong increase of HCN2 conductance, while p.(Ala363Val) and p.(Met374Leu) displayed dominant negative effects, leading to a partial loss of HCN2 channel function. By confocal imaging, we found that the p.(Leu377His), p.(Pro493Leu) and p.(Gly587Asp) pathogenic variants impaired membrane trafficking, resulting in a complete loss of HCN2 elicited currents in Xenopus oocytes. Structural 3D-analysis in depolarized and hyperpolarized states of HCN2 channels, revealed that the pathogenic variants p.(His205Gln), p.(Ser409Leu), p.(Arg324Cys), p.(Asn369Ser) and p.(Gly460Asp) modify molecular interactions altering HCN2 function. Taken together, our data broadens the clinical spectrum associated with HCN2 variants, and disclose that HCN2 is involved in developmental encephalopathy with or without epilepsy.

Segmentation of the C57BL/6J mouse cerebellum in magnetic resonance images.

The C57BL mouse is the centerpiece of efforts to use gene-targeting technology to understand cerebellar pathology, thus creating a need for a detailed magnetic resonance imaging (MRI) atlas of the cerebellum of this strain. In this study we present a methodology for systematic delineation of the vermal and hemispheric lobules of the C57BL/6J mouse cerebellum in magnetic resonance images. We have successfully delineated 38 cerebellar and cerebellar-related structures. The higher signal-to-noise ratio achieved by group averaging facilitated the identification of anatomical structures. In addition, we have calculated average region volumes and created probabilistic maps for each structure. The segmentation method and the probabilistic maps we have created will provide a foundation for future studies of cerebellar disorders using transgenic mouse models.

Antisense oligonucleotide therapy for KCNT1 encephalopathy.

Developmental and epileptic encephalopathies (DEEs) are characterized by pharmaco-resistant seizures with concomitant intellectual disability. Epilepsy of infancy with migrating focal seizures (EIMFS) is one of the most severe of these syndromes. De novo variants in ion channels, including gain-of-function variants in KCNT1, which encodes for sodium activated potassium channel protein KNa1.1, have been found to play a major role in the etiology of EIMFS. Here, we test a potential precision therapeutic approach in KCNT1-associated DEE using a gene-silencing antisense oligonucleotide (ASO) approach. We generated a mouse model carrying the KCNT1 p.P924L pathogenic variant; only the homozygous animals presented with the frequent, debilitating seizures and developmental compromise that are seen in patients. After a single intracerebroventricular bolus injection of a Kcnt1 gapmer ASO in symptomatic mice at postnatal day 40, seizure frequency was significantly reduced, behavioral abnormalities improved, and overall survival was extended compared with mice treated with a control ASO (nonhybridizing sequence). ASO administration at neonatal age was also well tolerated and effective in controlling seizures and extending the life span of treated animals. The data presented here provide proof of concept for ASO-based gene silencing as a promising therapeutic approach in KCNT1-associated epilepsies.

Segmentation of the mouse hippocampal formation in magnetic resonance images.

The hippocampal formation plays an important role in cognition, spatial navigation, learning, and memory. High resolution magnetic resonance (MR) imaging makes it possible to study in vivo changes in the hippocampus over time and is useful for comparing hippocampal volume and structure in wild type and mutant mice. Such comparisons demand a reliable way to segment the hippocampal formation. We have developed a method for the systematic segmentation of the hippocampal formation using the perfusion-fixed C57BL/6 mouse brain for application in longitudinal and comparative studies. Our aim was to develop a guide for segmenting over 40 structures in an adult mouse brain using 30 µm isotropic resolution images acquired with a 16.4 T MR imaging system and combined using super-resolution reconstruction.

Temperature elevation increases GABA(A) -mediated cortical inhibition in a mouse model of genetic epilepsy.

A missense mutation (R43Q) in the γ2 subunit of the γ-aminobutyric acid (GABA)(A) receptor is associated with generalized (genetic) epilepsy with febrile seizures plus (GEFS+). Heterozygous GABA(A) γ2(R43Q) mice displayed a lower temperature threshold for thermal seizures as compared to wild-type littermates. Temperature-dependent internalization of GABA(A) γ2(R43Q)-containing receptors has been proposed as a mechanism underlying febrile seizure genesis in patients with this mutation. We tested this idea using the GABA(A) γ2(R43Q) knockin mouse model and analyzed GABAergic miniature postsynaptic inhibitory currents (mIPSCs) in acute brain slices after exposure to varying temperatures. Incubation of slices at an elevated temperature increased mIPSC amplitude in neurons from heterozygous mice, with no change seen in wild-type controls. [³H]Flumazenil binding measured in whole-brain homogenates from mutant and control mice following elevation of body temperature showed no temperature-dependent differences in γ2-containing receptor density. Therefore, in vivo mouse data do not support earlier in vitro observations that proposed temperature-dependent internalization of γ2 R43Q containing GABA(A) receptors as the cellular mechanism underlying febrile seizure genesis in patients with the GABA(A) γ2(R43Q) mutation.

Atypical myelinogenesis and reduced axon caliber in the Scn1a variant model of Dravet syndrome: An electron microscopy pilot study of the developing and mature mouse corpus callosum.

Dravet Syndrome (DS) is a genetic neurodevelopmental disease. Recurrent severe seizures begin in infancy and co-morbidities follow, including developmental delay, cognitive and behavioral dysfunction. A majority of DS patients have an SCN1A heterozygous gene mutation. This mutation causes a loss-of-function in inhibitory neurons, initiating seizure onset. We have investigated whether the sodium channelopathy may result in structural changes in the DS model independent of seizures. Morphometric analyses of axons within the corpus callosum were completed at P16 and P50 in Scn1a heterozygote KO male mice and their age-matched wild-type littermates. Trainable machine learning algorithms were used to examine electron microscopy images of ~400 myelinated axons per animal, per genotype, including myelinated axon cross-section area, frequency distribution and g-ratios. Pilot data for Scn1a heterozygote KO mice demonstrate the average axon caliber was reduced in developing and adult mice. Qualitative analysis also shows micro-features marking altered myelination at P16 in the DS model, with myelin out-folding and myelin debris within phagocytic cells. The data has indicated, in the absence of behavioral seizures, factors that governed a shift toward small calibre axons at P16 have persisted in adult Scn1a heterozygote KO corpus callosum. The pilot study provides a basis for future meta-analysis that will enable robust estimates of the effects of the sodium channelopathy on axon architecture. We propose that early therapeutic strategies in DS could help minimize the effect of sodium channelopathies, beyond the impact of overt seizures, and therefore achieve better long-term treatment outcomes.

Antisense oligonucleotide therapy reduces seizures and extends life span in an SCN2A gain-of-function epilepsy model.

De novo variation in SCN2A can give rise to severe childhood disorders. Biophysical gain of function in SCN2A is seen in some patients with early seizure onset developmental and epileptic encephalopathy (DEE). In these cases, targeted reduction in SCN2A expression could substantially improve clinical outcomes. We tested this theory by central administration of a gapmer antisense oligonucleotide (ASO) targeting Scn2a mRNA in a mouse model of Scn2a early seizure onset DEE (Q/+ mice). Untreated Q/+ mice presented with spontaneous seizures at P1 and did not survive beyond P30. Administration of the ASO to Q/+ mice reduced spontaneous seizures and significantly extended life span. Across a range of behavioral tests, Scn2a ASO-treated Q/+ mice were largely indistinguishable from WT mice, suggesting treatment is well tolerated. A human SCN2A gapmer ASO could likewise impact the lives of patients with SCN2A gain-of-function DEE.

Heat opens axon initial segment sodium channels: a febrile seizure mechanism?

OBJECTIVE: A number of hypotheses have been put forward as to why humans respond to fever by seizing. The current leading hypotheses are that respiratory alkalosis produces an as yet unidentified change in neural excitability or that inflammatory mediators potentiate excitatory synaptic transmission. However, it is well known that ion channel gating rates increase with increased temperature. Furthermore, skeletal and cardiac sodium channel activation can be temperature sensitive in some situations. We measured the temperature sensitivity of the brain sodium channel, Na(V)1.2, to determine whether febrile temperatures might produce a direct increase in neuronal excitability. METHODS: The effect of temperature on Na(V)1.2 electrophysiological properties was measured in a transfected mammalian cell line. The subcellular location of Na(V)1.2 in the mouse brain was ascertained using antibodies against Na(V)1.2 and ankyrin-G. Computer simulation of a hippocampal granule cell model was used to predict the effect of temperature on action potential firing. RESULTS: As well as the expected increase in gating rates, the voltage dependence of activation became 7.6 mV more negative when the temperature was increased from 37 degrees C to 41 degrees C. Na(V)1.2 was localized to the axon initial segment in hippocampal and cortical neurons. Computer simulation showed that increased gating rates and the more negative activation dramatically increase neuronal excitability. INTERPRETATION: The direct effect of heat on ion channels localized to the site of action potential initiation potentially causes a profound increase in neuronal excitability. This is likely to contribute to febrile seizure genesis.

Using a Multiplex Nucleic Acid in situ Hybridization Technique to Determine HCN4 mRNA Expression in the Adult Rodent Brain.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels carry a non-selective cationic conductance, I h , which is important for modulating neuron excitability. Four genes (HCN1-4) encode HCN channels, with each gene having distinct expression and biophysical profiles. Here we use multiplex nucleic acid in situ hybridization to determine HCN4 mRNA expression within the adult mouse brain. We take advantage of this approach to detect HCN4 mRNA simultaneously with either HCN1 or HCN2 mRNA and markers of excitatory (VGlut-positive) and inhibitory (VGat-positive) neurons, which was not previously reported. We have developed a Fiji-based analysis code that enables quantification of mRNA expression within identified cell bodies. The highest HCN4 mRNA expression was found in the habenula (medial and lateral) and the thalamus. HCN4 mRNA was particularly high in the medial habenula with essentially no co-expression of HCN1 or HCN2 mRNA. An absence of I h -mediated "sag" in neurons recorded from the medial habenula of knockout mice confirmed that HCN4 channels are the predominant subtype in this region. Analysis in the thalamus revealed HCN4 mRNA in VGlut2-positive excitatory neurons that was always co-expressed with HCN2 mRNA. In contrast, HCN4 mRNA was undetectable in the nucleus reticularis. HCN4 mRNA expression was high in a subset of VGat-positive cells in the globus pallidus external. The majority of these neurons co-expressed HCN2 mRNA while a smaller subset also co-expressed HCN1 mRNA. In the striatum, a small subset of large cells which are likely to be giant cholinergic interneurons co-expressed high levels of HCN4 and HCN2 mRNA. The amygdala, cortex and hippocampus expressed low levels of HCN4 mRNA. This study highlights the heterogeneity of HCN4 mRNA expression in the brain and provides a morphological framework on which to better investigate the functional roles of HCN4 channels.

Abnormal Cell Sorting Underlies the Unique X-Linked Inheritance of PCDH19 Epilepsy.

X-linked diseases typically exhibit more severe phenotypes in males than females. In contrast, protocadherin 19 (PCDH19) mutations cause epilepsy in heterozygous females but spare hemizygous males. The cellular mechanism responsible for this unique pattern of X-linked inheritance is unknown. We show that PCDH19 contributes to adhesion specificity in a combinatorial manner such that mosaic expression of Pcdh19 in heterozygous female mice leads to striking sorting between cells expressing wild-type (WT) PCDH19 and null PCDH19 in the developing cortex, correlating with altered network activity. Complete deletion of PCDH19 in heterozygous mice abolishes abnormal cell sorting and restores normal network activity. Furthermore, we identify variable cortical malformations in PCDH19 epilepsy patients. Our results highlight the role of PCDH19 in determining cell adhesion affinities during cortical development and the way segregation of WT and null PCDH19 cells is associated with the unique X-linked inheritance of PCDH19 epilepsy.

Rare and common epilepsies converge on a shared gene regulatory network providing opportunities for novel antiepileptic drug discovery.

BACKGROUND: The relationship between monogenic and polygenic forms of epilepsy is poorly understood and the extent to which the genetic and acquired epilepsies share common pathways is unclear. Here, we use an integrated systems-level analysis of brain gene expression data to identify molecular networks disrupted in epilepsy. RESULTS: We identified a co-expression network of 320 genes (M30), which is significantly enriched for non-synonymous de novo mutations ascertained from patients with monogenic epilepsy and for common variants associated with polygenic epilepsy. The genes in the M30 network are expressed widely in the human brain under tight developmental control and encode physically interacting proteins involved in synaptic processes. The most highly connected proteins within the M30 network were preferentially disrupted by deleterious de novo mutations for monogenic epilepsy, in line with the centrality-lethality hypothesis. Analysis of M30 expression revealed consistent downregulation in the epileptic brain in heterogeneous forms of epilepsy including human temporal lobe epilepsy, a mouse model of acquired temporal lobe epilepsy, and a mouse model of monogenic Dravet (SCN1A) disease. These results suggest functional disruption of M30 via gene mutation or altered expression as a convergent mechanism regulating susceptibility to epilepsy broadly. Using the large collection of drug-induced gene expression data from Connectivity Map, several drugs were predicted to preferentially restore the downregulation of M30 in epilepsy toward health, most notably valproic acid, whose effect on M30 expression was replicated in neurons. CONCLUSIONS: Taken together, our results suggest targeting the expression of M30 as a potential new therapeutic strategy in epilepsy.

Sodium channel ß1 subunit localizes to axon initial segments of excitatory and inhibitory neurons and shows regional heterogeneity in mouse brain.

The ß1 subunit of voltage-gated sodium channels, Nav ß1, plays multiple roles in neurons spanning electrophysiological modulation of sodium channel α subunits to cell adhesion and neurite outgrowth. This study used immunohistochemistry to investigate Nav ß1 subneuronal and regional expression. Nav ß1 was enriched at axon initial segments (AIS) and nodes of Ranvier. Nav ß1 expression at the AIS was detected throughout the brain, predominantly in the hippocampus, cortex, and cerebellum. Despite expression of Nav ß1 in both excitatory and inhibitory AIS, it displayed a marked and fine-grained heterogeneity of expression. Such heterogeneity could have important implications for the tuning of single neuronal and regional excitability, especially in view of the fact that Nav ß1 coexpressed with Nav 1.1, Nav 1.2, and Nav 1.6 subunits. The disruption of Nav ß1 AIS expression by a human epilepsy-causing C121W genetic mutation in Nav ß1 was also investigated using a mouse model. AIS expression of Nav ß1 was reduced by approximately 50% in mice heterozygous for the C121W mutation and was abolished in homozygotes, suggesting that loss of Nav α subunit modulation by Nav ß1 contributes to the mechanism of epileptogenesis in these animals as well as in patients.