Live imaging of excitable axonal microdomains in ankyrin-G-GFP mice.

The axon initial segment (AIS) constitutes not only the site of action potential initiation, but also a hub for activity-dependent modulation of output generation. Recent studies shedding light on AIS function used predominantly post-hoc approaches since no robust murine in vivo live reporters exist. Here, we introduce a reporter line in which the AIS is intrinsically labeled by an ankyrin-G-GFP fusion protein activated by Cre recombinase, tagging the native Ank3 gene. Using confocal, superresolution, and two-photon microscopy as well as whole-cell patch-clamp recordings in vitro, ex vivo, and in vivo, we confirm that the subcellular scaffold of the AIS and electrophysiological parameters of labeled cells remain unchanged. We further uncover rapid AIS remodeling following increased network activity in this model system, as well as highly reproducible in vivo labeling of AIS over weeks. This novel reporter line allows longitudinal studies of AIS modulation and plasticity in vivo in real-time and thus provides a unique approach to study subcellular plasticity in a broad range of applications.

Physical and functional convergence of the autism risk genes Scn2a and Ank2 in neocortical pyramidal cell dendrites.

Dysfunction in sodium channels and their ankyrin scaffolding partners have both been implicated in neurodevelopmental disorders, including autism spectrum disorder (ASD). In particular, the genes SCN2A, which encodes the sodium channel NaV1.2, and ANK2, which encodes ankyrin-B, have strong ASD association. Recent studies indicate that ASD-associated haploinsufficiency in Scn2a impairs dendritic excitability and synaptic function in neocortical pyramidal cells, but how NaV1.2 is anchored within dendritic regions is unknown. Here, we show that ankyrin-B is essential for scaffolding NaV1.2 to the dendritic membrane of mouse neocortical neurons and that haploinsufficiency of Ank2 phenocopies intrinsic dendritic excitability and synaptic deficits observed in Scn2a+/- conditions. These results establish a direct, convergent link between two major ASD risk genes and reinforce an emerging framework suggesting that neocortical pyramidal cell dendritic dysfunction can contribute to neurodevelopmental disorder pathophysiology.

Inhibitory synaptic transmission is impaired in the Kölliker-Fuse of male, but not female, Rett syndrome mice.

Rett syndrome (RTT) is a severe neurodevelopmental disorder that mainly affects females due to silencing mutations in the X-linked MECP2 gene. One of the most troubling symptoms of RTT is breathing irregularity, including apneas, breath-holds, and hyperventilation. Mice with silencing mutations in Mecp2 exhibit breathing abnormalities similar to human patients and serve as useful models for studying mechanisms underlying breathing problems in RTT. Previous work implicated the pontine, respiratory-controlling Kölliker-Fuse (KF) in the breathing problems in RTT. The goal of this study was to test the hypothesis that inhibitory synaptic transmission is deficient in KF neurons from symptomatic male and female RTT mice. We performed whole cell voltage-clamp recordings from KF neurons in acute brain slices to examine spontaneous and electrically evoked inhibitory post-synaptic currents (IPSCs) in RTT mice and age- and sex-matched wild-type mice. The frequency of spontaneous IPSCs was reduced in KF neurons from male RTT mice but surprisingly not in female RTT mice. In addition, electrically evoked IPSCs were less reliable in KF neurons from male, but not female, RTT mice, which was positively correlated with paired-pulse facilitation, indicating decreased probability of release. KF neurons from male RTT mice were also more excitable and exhibited shorter-duration action potentials. Increased excitability of KF neurons from male mice was not explained by changes in axon initial segment length. These findings indicate impaired inhibitory neurotransmission and increased excitability of KF neurons in male but not female RTT mice and suggest that sex-dependent mechanisms contribute to breathing problems in RTT.NEW & NOTEWORTHY Kölliker-Fuse (KF) neurons in acute brain slices from male Rett syndrome (RTT) mice receive reduced inhibitory synaptic inputs compared with wild-type littermates. In female RTT mice, inhibitory transmission was not different in KF neurons compared with controls. The results from this study show that sex-specific alterations in synaptic transmission occur in the KF of RTT mice.

Effects of chronic lithium treatment on neuronal excitability and GABAergic transmission in an Ank3 mutant mouse model.

Bipolar disorder (BD) is a common psychiatric disease that can lead to psychosocial disability, decreased quality of life, and high risk for suicide. Genome-wide association studies have shown that the ANK3 gene is a significant risk factor for BD, but the mechanisms involved in BD pathophysiology are not yet fully understood. Previous work has shown that ankyrin-G, the protein encoded by ANK3, stabilizes inhibitory synapses in vivo through its interaction with the GABAA receptor-associated protein (GABARAP). We generated a mouse model with a missense p.W1989R mutation in Ank3, that abolishes the interaction between ankyrin-G and GABARAP, which leads to reduced inhibitory signaling in the somatosensory cortex and increased pyramidal cell excitability. Humans with the same mutation exhibit BD symptoms, which can be attenuated with lithium therapy. In this study, we describe that chronic treatment of Ank3 p.W1989R mice with lithium normalizes neuronal excitability in cortical pyramidal neurons and increases inhibitory GABAergic postsynaptic currents. The same outcome in inhibitory transmission was observed when mice were treated with the GSK-3ß inhibitor Tideglusib. These results suggest that lithium treatment modulates the excitability of pyramidal neurons in the cerebral cortex by increasing GABAergic neurotransmission, likely via GSK-3 inhibition. In addition to the importance of these findings regarding ANK3 variants as a risk factor for BD development, this study may have significant implications for treating other psychiatric disorders associated with alterations in inhibitory signaling, such as schizophrenia, autism spectrum disorder, and major depressive disorder.

A Novel Approach to Study Coherent γ-Band Oscillations in Hippocampal Brain Sections.

γ-Band oscillations (GBOs) are generated by fast-spiking interneurons (FSIs) and are critical for cognitive functions. Abnormalities in GBOs are frequently observed in schizophrenia and bipolar disorder and are strongly correlated with cognitive impairment. However, the underlying mechanisms are poorly understood. Studying GBOs in ex vivo preparations is challenging because of high energy demands and the need for continuous oxygen delivery to the tissue. As a result, GBOs are typically studied in brain tissue from very young animals or in experimental setups that maximize oxygen supply but compromise spatial resolution. Thus, there is a limited understanding of how GBOs interact within and between different brain structures and in brain tissue from mature animals. To address these limitations, we have developed a novel approach for studying GBOs in ex vivo hippocampal slices from mature animals, using 60-channel, perforated microelectrode arrays (pMEAs). pMEAs enhance oxygen delivery and increase spatial resolution in electrophysiological recordings, enabling comprehensive analyses of GBO synchronization within discrete brain structures. We found that transecting the Schaffer collaterals, a neural pathway within the hippocampus, impairs GBO coherence between CA1 and CA3 subfields. Furthermore, we validated our approach by studying GBO coherence in an Ank3 mutant mouse model exhibiting inhibitory synaptic dysfunction. We discovered that GBO coherence remains intact in the CA3 subfield of these mutant mice but is impaired within and between the CA1 subfield. Overall, our approach offers significant potential to characterize GBOs in ex vivo brain sections of animal models, enhancing our understanding of network dysfunction in psychiatric disorders.

Extracellular Tau Oligomers Damage the Axon Initial Segment.

BACKGROUND: In Alzheimer's disease (AD) brain, neuronal polarity and synaptic connectivity are compromised. A key structure for regulating polarity and functions of neurons is the axon initial segment (AIS), which segregates somatodendritic from axonal proteins and initiates action potentials. Toxic tau species, including extracellular oligomers (xcTauOs), spread tau pathology from neuron to neuron by a prion-like process, but few other cell biological effects of xcTauOs have been described. OBJECTIVE: Test the hypothesis that AIS structure is sensitive to xcTauOs. METHODS: Cultured wild type (WT) and tau knockout (KO) mouse cortical neurons were exposed to xcTauOs, and quantitative western blotting and immunofluorescence microscopy with anti-TRIM46 monitored effects on the AIS. The same methods were used to compare TRIM46 and two other resident AIS proteins in human hippocampal tissue obtained from AD and age-matched non-AD donors. RESULTS: Without affecting total TRIM46 levels, xcTauOs reduce the concentration of TRIM46 within the AIS and cause AIS shortening in cultured WT, but not TKO neurons. Lentiviral-driven tau expression in tau KO neurons rescues AIS length sensitivity to xcTauOs. In human AD hippocampus, the overall protein levels of multiple resident AIS proteins are unchanged compared to non-AD brain, but TRIM46 concentration within the AIS and AIS length are reduced in neurons containing neurofibrillary tangles. CONCLUSION: xcTauOs cause partial AIS damage in cultured neurons by a mechanism dependent on intracellular tau, thereby raising the possibility that the observed AIS reduction in AD neurons in vivo is caused by xcTauOs working in concert with endogenous neuronal tau.

Ankyrin-B is lipid-modified by S-palmitoylation to promote dendritic membrane scaffolding of voltage-gated sodium channel NaV1.2 in neurons.

Neuronal ankyrin-B is an intracellular scaffolding protein that plays multiple roles in the axon. By contrast, relatively little is known about the function of ankyrin-B in dendrites, where ankyrin-B is also localized in mature neurons. Recently, we showed that ankyrin-B acts as a scaffold for the voltage-gated sodium channel, NaV1.2, in dendrites of neocortical pyramidal neurons. How ankyrin-B is itself targeted to the dendritic membrane is not well understood. Here, we report that ankyrin-B is lipid-modified by S-palmitoylation to promote dendritic localization of NaV1.2. We identify the palmitoyl acyl transferase zDHHC17 as a key mediator of ankyrin-B palmitoylation in heterologous cells and in neurons. Additionally, we find that zDHHC17 regulates ankyrin-B protein levels independently of its S-acylation function through a conserved binding mechanism between the ANK repeat domain of zDHHC17 and the zDHHC ankyrin-repeat binding motif of ankyrin-B. We subsequently identify five cysteines in the N-terminal ankyrin repeat domain of ankyrin-B that are necessary for ankyrin-B palmitoylation. Mutation of these five cysteines to alanines not only abolishes ankyrin-B palmitoylation, but also prevents ankyrin-B from scaffolding NaV1.2 at dendritic membranes of neurons due to ankyrin-B's inability to localize properly at dendrites. Thus, we show palmitoylation is critical for localization and function of ankyrin-B at dendrites. Strikingly, loss of ankyrin-B palmitoylation does not affect ankyrin-B-mediated axonal cargo transport of synaptic vesicle synaptotagmin-1 in neurons. This is the first demonstration of S-palmitoylation of ankyrin-B as an underlying mechanism required for ankyrin-B localization and function in scaffolding NaV1.2 at dendrites.

DSCAM gene triplication causes excessive GABAergic synapses in the neocortex in Down syndrome mouse models.

Down syndrome (DS) is caused by the trisomy of human chromosome 21 (HSA21). A major challenge in DS research is to identify the HSA21 genes that cause specific symptoms. Down syndrome cell adhesion molecule (DSCAM) is encoded by a HSA21 gene. Previous studies have shown that the protein level of the Drosophila homolog of DSCAM determines the size of presynaptic terminals. However, whether the triplication of DSCAM contributes to presynaptic development in DS remains unknown. Here, we show that DSCAM levels regulate GABAergic synapses formed on neocortical pyramidal neurons (PyNs). In the Ts65Dn mouse model for DS, where DSCAM is overexpressed due to DSCAM triplication, GABAergic innervation of PyNs by basket and chandelier interneurons is increased. Genetic normalization of DSCAM expression rescues the excessive GABAergic innervations and the increased inhibition of PyNs. Conversely, loss of DSCAM impairs GABAergic synapse development and function. These findings demonstrate excessive GABAergic innervation and synaptic transmission in the neocortex of DS mouse models and identify DSCAM overexpression as the cause. They also implicate dysregulated DSCAM levels as a potential pathogenic driver in related neurological disorders.

Synaptotagmin-7 facilitates acetylcholine release in splanchnic nerve-chromaffin cell synapses during nerve activity.

Disturbances that threaten homeostasis elicit activation of the sympathetic nervous system (SNS) and the adrenal medulla. The effectors discharge as a unit to drive global and immediate changes in whole-body physiology. Descending sympathetic information is conveyed to the adrenal medulla via preganglionic splanchnic fibers. These fibers pass into the gland and synapse onto chromaffin cells, which synthesize, store, and secrete catecholamines and vasoactive peptides. While the importance of the sympatho-adrenal branch of the autonomic nervous system has been appreciated for many decades, the mechanisms underlying transmission between presynaptic splanchnic neurons and postsynaptic chromaffin cells have remained obscure. In contrast to chromaffin cells, which have enjoyed sustained attention as a model system for exocytosis, even the Ca2+ sensors that are expressed within splanchnic terminals have not yet been identified. This study shows that a ubiquitous Ca2+-binding protein, synaptotagmin-7 (Syt7), is expressed within the fibers that innervate the adrenal medulla, and that its absence can alter synaptic transmission in the preganglionic terminals of chromaffin cells. The prevailing impact in synapses that lack Syt7 is a decrease in synaptic strength and neuronal short-term plasticity. Evoked excitatory postsynaptic currents (EPSCs) in Syt7 KO preganglionic terminals are smaller in amplitude than in wild-type synapses stimulated in an identical manner. Splanchnic inputs also display robust short-term presynaptic facilitation, which is compromised in the absence of Syt7. These data reveal, for the first time, a role for any synaptotagmin at the splanchnic-chromaffin cell synapse. They also suggest that Syt7 has actions at synaptic terminals that are conserved across central and peripheral branches of the nervous system.

Sodium channel ß1 subunits are post-translationally modified by tyrosine phosphorylation, S-palmitoylation, and regulated intramembrane proteolysis.

Voltage-gated sodium channel (VGSC) ß1 subunits are multifunctional proteins that modulate the biophysical properties and cell-surface localization of VGSC α subunits and participate in cell-cell and cell-matrix adhesion, all with important implications for intracellular signal transduction, cell migration, and differentiation. Human loss-of-function variants in SCN1B, the gene encoding the VGSC ß1 subunits, are linked to severe diseases with high risk for sudden death, including epileptic encephalopathy and cardiac arrhythmia. We showed previously that ß1 subunits are post-translationally modified by tyrosine phosphorylation. We also showed that ß1 subunits undergo regulated intramembrane proteolysis via the activity of ß-secretase 1 and γ-secretase, resulting in the generation of a soluble intracellular domain, ß1-ICD, which modulates transcription. Here, we report that ß1 subunits are phosphorylated by FYN kinase. Moreover, we show that ß1 subunits are S-palmitoylated. Substitution of a single residue in ß1, Cys-162, to alanine prevented palmitoylation, reduced the level of ß1 polypeptides at the plasma membrane, and reduced the extent of ß1-regulated intramembrane proteolysis, suggesting that the plasma membrane is the site of ß1 proteolytic processing. Treatment with the clathrin-mediated endocytosis inhibitor, Dyngo-4a, re-stored the plasma membrane association of ß1-p.C162A to WT levels. Despite these observations, palmitoylation-null ß1-p.C162A modulated sodium current and sorted to detergent-resistant membrane fractions normally. This is the first demonstration of S-palmitoylation of a VGSC ß subunit, establishing precedence for this post-translational modification as a regulatory mechanism in this protein family.

Palmitoylation: A Fatty Regulator of Myocardial Electrophysiology.

Regulation of cardiac physiology is well known to occur through the action of kinases that reversibly phosphorylate ion channels, calcium handling machinery, and signaling effectors. However, it is becoming increasingly apparent that palmitoylation or S-acylation, the post-translational modification of cysteines with saturated fatty acids, plays instrumental roles in regulating the localization, activity, stability, sorting, and function of numerous proteins, including proteins known to have essential functions in cardiomyocytes. However, the impact of this modification on cardiac physiology requires further investigation. S-acylation is catalyzed by the zDHHC family of S-acyl transferases that localize to intracellular organelle membranes or the sarcolemma. Recent work has begun to uncover functions of S-acylation in the heart, particularly in the regulation of cardiac electrophysiology, including modification of the sodium-calcium exchanger, phospholemman and the cardiac sodium pump, as well as the voltage-gated sodium channel. Elucidating the regulatory functions of zDHHC enzymes in cardiomyocytes and determination of how S-acylation is altered in the diseased heart will shed light on how these modifications participate in cardiac pathogenesis and potentially identify novel targets for the treatment of cardiovascular disease. Indeed, proteins with critical signaling roles in the heart are also S-acylated, including receptors and G-proteins, yet the dynamics and functions of these modifications in myocardial physiology have not been interrogated. Here, we will review what is known about zDHHC enzymes and substrate S-acylation in myocardial physiology and highlight future areas of investigation that will uncover novel functions of S-acylation in cardiac homeostasis and pathophysiology.

Synaptotagmin-7 enhances calcium-sensing of chromaffin cell granules and slows discharge of granule cargos.

Synaptotagmin-7 (Syt-7) is one of two major calcium sensors for exocytosis in adrenal chromaffin cells, the other being synaptotagmin-1 (Syt-1). Despite a broad appreciation for the importance of Syt-7, questions remain as to its localization, function in mediating discharge of dense core granule cargos, and role in triggering release in response to physiological stimulation. These questions were addressed using two distinct experimental preparations-mouse chromaffin cells lacking endogenous Syt-7 (KO cells) and a reconstituted system employing cell-derived granules expressing either Syt-7 or Syt-1. First, using immunofluorescence imaging and subcellular fractionation, it is shown that Syt-7 is widely distributed in organelles, including dense core granules. Total internal reflection fluorescence (TIRF) imaging demonstrates that the kinetics and probability of granule fusion in Syt-7 KO cells stimulated by a native secretagogue, acetylcholine, are markedly lower than in WT cells. When fusion is observed, fluorescent cargo proteins are discharged more rapidly when only Syt-1 is available to facilitate release. To determine the extent to which the aforementioned results are attributable purely to Syt-7, granules expressing only Syt-7 or Syt-1 were triggered to fuse on planar supported bilayers bearing plasma membrane SNARE proteins. Here, as in cells, Syt-7 confers substantially greater calcium sensitivity to granule fusion than Syt-1 and slows the rate at which cargos are released. Overall, this study demonstrates that by virtue of its high affinity for calcium and effects on fusion pore expansion, Syt-7 plays a central role in regulating secretory output from adrenal chromaffin cells.

Spatial organization of palmitoyl acyl transferases governs substrate localization and function.

Protein palmitoylation is a critical posttranslational modification that regulates protein trafficking, localization, stability, sorting and function. In mammals, addition of this lipid modification onto proteins is mediated by a family of 23 palmitoyl acyl transferases (PATs). PATs often palmitoylate substrates in a promiscuous manner, precluding our understanding of how these enzymes achieve specificity for their substrates. Despite generous efforts to identify consensus motifs defining PAT-substrate specificity, it remains to be determined whether additional factors beyond interaction motifs, such as local palmitoylation, participate in PAT-substrate selection. In this review, we emphasize the role of local palmitoylation, in which substrates are palmitoylated and trapped in the same subcellular compartments as their PATs, as a mechanism of enzyme-substrate specificity. We focus here on non-Golgi-localized PATs, as physical proximity to their substrates enables them to engage in local palmitoylation, compared to Golgi PATs, which often direct trafficking of their substrates elsewhere. PAT subcellular localization may be an under-recognized, yet important determinant of PAT-substrate specificity that may work in conjunction or completely independently of interaction motifs. We also discuss some current hypotheses about protein motifs that contribute to localization of non-Golgi-localized PATs, important for the downstream targeting of their substrates.

Critical roles of αII spectrin in brain development and epileptic encephalopathy.

The nonerythrocytic α-spectrin-1 (SPTAN1) gene encodes the cytoskeletal protein αII spectrin. Mutations in SPTAN1 cause early infantile epileptic encephalopathy type 5 (EIEE5); however, the role of αII spectrin in neurodevelopment and EIEE5 pathogenesis is unknown. Prior work suggests that αII spectrin is absent in the axon initial segment (AIS) and contributes to a diffusion barrier in the distal axon. Here, we have shown that αII spectrin is expressed ubiquitously in rodent and human somatodendritic and axonal domains. CRISPR-mediated deletion of Sptan1 in embryonic rat forebrain by in utero electroporation caused altered dendritic and axonal development, loss of the AIS, and decreased inhibitory innervation. Overexpression of human EIEE5 mutant SPTAN1 in embryonic rat forebrain and mouse hippocampal neurons led to similar developmental defects that were also observed in EIEE5 patient-derived neurons. Additionally, patient-derived neurons displayed aggregation of spectrin complexes. Taken together, these findings implicate αII spectrin in critical aspects of dendritic and axonal development and synaptogenesis, and support a dominant-negative mechanism of SPTAN1 mutations in EIEE5.

Genetic disruption of ankyrin-G in adult mouse forebrain causes cortical synapse alteration and behavior reminiscent of bipolar disorder.

Genome-wide association studies have implicated the ANK3 locus in bipolar disorder, a major human psychotic illness. ANK3 encodes ankyrin-G, which organizes the neuronal axon initial segment (AIS). We generated a mouse model with conditional disruption of ANK3 in pyramidal neurons of the adult forebrain (Ank-G cKO). This resulted in the expected loss of pyramidal neuron AIS voltage-gated sodium and potassium channels. There was also dramatic loss of markers of afferent GABAergic cartridge synapses, resembling the cortical microcircuitry changes in brains from psychotic patients, and suggesting disinhibition. Expression of c-fos was increased in cortical pyramidal neurons, consistent with increased neuronal activity due to disinhibition. The mice showed robust behavioral phenotypes reminiscent of aspects of human mania, ameliorated by antimania drugs lithium and valproate. Repeated social defeat stress resulted in repeated episodes of dramatic behavioral changes from hyperactivity to "depression-like" behavior, suggestive of some aspects of human bipolar disorder. Overall, we suggest that this Ank-G cKO mouse model recapitulates some of the core features of human bipolar disorder and indicates that cortical microcircuitry alterations during adulthood may be involved in pathogenesis. The model may be useful for studying disease pathophysiology and for developing experimental therapeutics.

Synaptotagmin isoforms confer distinct activation kinetics and dynamics to chromaffin cell granules.

Adrenomedullary chromaffin cells respond to sympathetic nervous system activation by secreting a cocktail of potent neuropeptides and hormones into the circulation. The distinct phases of the chromaffin cell secretory response have been attributed to the progressive fusion of distinct populations of dense core granules with different activation kinetics. However, it has been difficult to define what distinguishes these populations at the molecular level. Functional segregation of granule pools may depend on selective sorting of synaptotagmin-1 (Syt-1) and synaptotagmin-7 (Syt-7), which our previous work showed are rarely cosorted to the same granule. Here we assess the consequences of selective sorting of Syt isoforms in chromaffin cells, particularly with respect to granule dynamics and activation kinetics. Upon depolarization of cells expressing fluorescent Syt isoforms using elevated K+, we find that Syt-7 granules fuse with faster kinetics than Syt-1 granules, irrespective of stimulation strength. Pharmacological blockade of Ca2+ channels reveals differential dependence of Syt-1 versus Syt-7 granule exocytosis on Ca2+ channel subtypes. Syt-7 granules also show a greater tendency to fuse in clusters than Syt-1 granules, and granules harboring Syt-1 travel a greater distance before fusion than those with Syt-7, suggesting that there is spatial and fusion-site heterogeneity among the two granule populations. However, the greatest functional difference between granule populations is their responsiveness to Ca2+ Upon introduction of Ca2+ into permeabilized cells, Syt-7 granules fuse with fast kinetics and high efficacy, even at low Ca2+ levels (e.g., when cells are weakly stimulated). Conversely, Syt-1 granules require a comparatively larger increase in intracellular Ca2+ for activation. At Ca2+ concentrations above 30 µM, activation kinetics are faster for Syt-1 granules than for Syt-7 granules. Our study provides evidence for functional specialization of chromaffin cell granules via selective expression of Syt isoforms with different Ca2+ sensitivities.

Chemoselective ratiometric imaging of protein S-sulfenylation.

Here we report a ratiometric fluorescent probe for chemoselective conjugation to sulfenic acids in living cells. Our approach couples an α-fluoro-substituted dimedone to an aminonaphthalene fluorophore (F-DiNap), which upon sulfenic acid conjugation is locked as the 1,3-diketone, changing the fluorophore excitation. F-DiNap reacts with S-sulfenylated proteins at equivalent rates to current probes, but the α-fluorine substitution blocks side-reactions with biological aldehydes.

Axonal Membranes and Their Domains: Assembly and Function of the Axon Initial Segment and Node of Ranvier.

Neurons are highly specialized cells of the nervous system that receive, process and transmit electrical signals critical for normal brain function. Here, we review the intricate organization of axonal membrane domains that facilitate rapid action potential conduction underlying communication between complex neuronal circuits. Two critical excitable domains of vertebrate axons are the axon initial segment (AIS) and the nodes of Ranvier, which are characterized by the high concentrations of voltage-gated ion channels, cell adhesion molecules and specialized cytoskeletal networks. The AIS is located at the proximal region of the axon and serves as the site of action potential initiation, while nodes of Ranvier, gaps between adjacent myelin sheaths, allow rapid propagation of the action potential through saltatory conduction. The AIS and nodes of Ranvier are assembled by ankyrins, spectrins and their associated binding partners through the clustering of membrane proteins and connection to the underlying cytoskeleton network. Although the AIS and nodes of Ranvier share similar protein composition, their mechanisms of assembly are strikingly different. Here we will cover the mechanisms of formation and maintenance of these axonal excitable membrane domains, specifically highlighting the similarities and differences between them. We will also discuss recent advances in super resolution fluorescence imaging which have elucidated the arrangement of the submembranous axonal cytoskeleton revealing a surprising structural organization necessary to maintain axonal organization and function. Finally, human mutations in axonal domain components have been associated with a growing number of neurological disorders including severe cognitive dysfunction, epilepsy, autism, neurodegenerative diseases and psychiatric disorders. Overall, this review highlights the assembly, maintenance and function of axonal excitable domains, particularly the AIS and nodes of Ranvier, and how abnormalities in these processes may contribute to disease.