Impact on backpropagation of the spatial heterogeneity of sodium channel kinetics in the axon initial segment.

In a variety of neurons, action potentials (APs) initiate at the proximal axon, within a region called the axon initial segment (AIS), which has a high density of voltage-gated sodium channels (NaVs) on its membrane. In pyramidal neurons, the proximal AIS has been reported to exhibit a higher proportion of NaVs with gating properties that are "right-shifted" to more depolarized voltages, compared to the distal AIS. Further, recent experiments have revealed that as neurons develop, the spatial distribution of NaV subtypes along the AIS can change substantially, suggesting that neurons tune their excitability by modifying said distribution. When neurons are stimulated axonally, computational modelling has shown that this spatial separation of gating properties in the AIS enhances the backpropagation of APs into the dendrites. In contrast, in the more natural scenario of somatic stimulation, our simulations show that the same distribution can impede backpropagation, suggesting that the choice of orthodromic versus antidromic stimulation can bias or even invert experimental findings regarding the role of NaV subtypes in the AIS. We implemented a range of hypothetical NaV distributions in the AIS of three multicompartmental pyramidal cell models and investigated the precise kinetic mechanisms underlying such effects, as the spatial distribution of NaV subtypes is varied. With axonal stimulation, proximal NaV availability dominates, such that concentrating right-shifted NaVs in the proximal AIS promotes backpropagation. However, with somatic stimulation, the models are insensitive to availability kinetics. Instead, the higher activation threshold of right-shifted NaVs in the AIS impedes backpropagation. Therefore, recently observed developmental changes to the spatial separation and relative proportions of NaV1.2 and NaV1.6 in the AIS differentially impact activation and availability. The observed effects on backpropagation, and potentially learning via its putative role in synaptic plasticity (e.g. through spike-timing-dependent plasticity), are opposite for orthodromic versus antidromic stimulation, which should inform hypotheses about the impact of the developmentally regulated subcellular localization of these NaV subtypes.

Analysis of the effect of the scorpion toxin AaH-II on action potential generation in the axon initial segment.

The toxin AaH-II, from the scorpion Androctonus australis Hector venom, is a 64 amino acid peptide that targets voltage-gated Na+ channels (VGNCs) and slows their inactivation. While at macroscopic cellular level AaH-II prolongs the action potential (AP), a functional analysis of the effect of the toxin in the axon initial segment (AIS), where VGNCs are highly expressed, was never performed so far. Here, we report an original analysis of the effect of AaH-II on the AP generation in the AIS of neocortical layer-5 pyramidal neurons from mouse brain slices. After determining that AaH-II does not discriminate between Nav1.2 and Nav1.6, i.e. between the two VGNC isoforms expressed in this neuron, we established that 7 nM was the smallest toxin concentration producing a minimal detectable deformation of the somatic AP after local delivery of the toxin. Using membrane potential imaging, we found that, at this minimal concentration, AaH-II substantially widened the AP in the AIS. Using ultrafast Na+ imaging, we found that local application of 7 nM AaH-II caused a large increase in the slower component of the Na+ influx in the AIS. Finally, using ultrafast Ca2+ imaging, we observed that 7 nM AaH-II produces a spurious slow Ca2+ influx via Ca2+-permeable VGNCs. Molecules targeting VGNCs, including peptides, are proposed as potential therapeutic tools. Thus, the present analysis in the AIS can be considered a general proof-of-principle on how high-resolution imaging techniques can disclose drug effects that cannot be observed when tested at the macroscopic level.

Evidence for glia-mediated, age-dependent remodeling of myelin at the axon initial segment.

Due to its proximity to the axon initial segment (AIS), the paranode of the first myelin segment can influence the threshold for action potentials and how a neuron participates in a neuronal circuit. Using serial section electron microscopy, we examined its three-dimensional (3D) organization in the ventral horn of the mouse spinal cord. The myelin loops of postnatal day 18 mice resemble those at the node of Ranvier. However, in 3-month-old mice, 13 of 22 para-AIS showed 4 types of alteration: (A) A cytoplasmic foot process, with ultrastructural characteristics of an astrocyte, was interposed between the axolemma and the myelin loops. (B) A thin extension of the inner tongue was present between the foot process and axolemma. (C) The foot process was absent. The inner tongue extension was a broad lamella from which a thin extension reached beyond the loops and spiraled around axon. (D) One set of loops was adjacent to the axon, and another was further back and underlain by compact myelin. We suggest that (A)-(C) are steps in a progression toward (D). In this progression, a glial process displaces the original loops, the inner tongue reactivates and extends beneath the foot process, then wraps around the axon to form a new set of loops. This is the first study of the 3D organization of myelin at the AIS and provides evidence for glia-mediated age-dependent remodeling at this critical region.

Static magnetic stimulation induces structural plasticity at the axon initial segment of inhibitory cortical neurons.

Static magnetic stimulation (SMS) is a form of non-invasive brain stimulation that alters neural activity and induces neural plasticity that outlasts the period of stimulation. This can modify corticospinal excitability or motor behaviours, suggesting that SMS may alter the intrinsic excitability of neurons. In mammalian neurons, the axon initial segment (AIS) is the site of action potential initiation and undergoes structural plasticity (changes in length and position from the soma) as a homeostatic mechanism to counteract chronic changes in neuronal activity. We investigated whether the chronic application of SMS (6 and 48 h, 0.5 T) induces structural AIS plasticity in postnatally derived primary cortical neurons. Following 6 h of SMS, we observed a shortening in mean AIS length compared to control, that persisted 24 h post stimulation. In contrast, 48 h of SMS induced an immediate distal shift that persisted 24 h post-stimulation. Pharmacological blockade of voltage gated L/T-type calcium channels during stimulation did not prevent SMS-induced AIS structural plasticity. Our findings provide the foundation to expand the use of chronic SMS as a non-invasive method to promote AIS plasticity.

Concussive Head Trauma Deranges Axon Initial Segment Function in Axotomized and Intact Layer 5 Pyramidal Neurons.

The axon initial segment (AIS) is a critical locus of control of action potential (AP) generation and neuronal information synthesis. Concussive traumatic brain injury gives rise to diffuse axotomy, and the majority of neocortical axonal injury arises at the AIS. Consequently, concussive traumatic brain injury might profoundly disrupt the functional specialization of this region. To investigate this hypothesis, one and two days after mild central fluid percussion injury in Thy1-YFP-H mice, we recorded high-resolution APs from axotomized and adjacent intact layer 5 pyramidal neurons and applied a second derivative (2o) analysis to measure the AIS- and soma-regional contributions to the AP upstroke. All layer 5 pyramidal neurons recorded from sham animals manifested two stark 2o peaks separated by a negative intervening slope. In contrast, within injured mice, we discovered a subset of axotomized layer 5 pyramidal neurons in which the AIS-regional 2o peak was abolished, a functional perturbation associated with diminished excitability, axonal sprouting and distention of the AIS as assessed by staining for ankyrin-G. Our analysis revealed an additional subpopulation of both axotomized and intact layer 5 pyramidal neurons that manifested a melding together of the AIS- and soma-regional 2o peaks, suggesting a more subtle aberration of sodium channel function and/or translocation of the AIS initiation zone closer to the soma. When these experiments were repeated in animals in which cyclophilin-D was knocked out, these effects were ameliorated, suggesting that trauma-induced AIS functional perturbation is associated with mitochondrial calcium dysregulation.

Immunoproximity biotinylation reveals the axon initial segment proteome.

The axon initial segment (AIS) is a specialized neuronal compartment required for action potential generation and neuronal polarity. However, understanding the mechanisms regulating AIS structure and function has been hindered by an incomplete knowledge of its molecular composition. Here, using immuno-proximity biotinylation we further define the AIS proteome and its dynamic changes during neuronal maturation. Among the many AIS proteins identified, we show that SCRIB is highly enriched in the AIS both in vitro and in vivo, and exhibits a periodic architecture like the axonal spectrin-based cytoskeleton. We find that ankyrinG interacts with and recruits SCRIB to the AIS. However, loss of SCRIB has no effect on ankyrinG. This powerful and flexible approach further defines the AIS proteome and provides a rich resource to elucidate the mechanisms regulating AIS structure and function.

Thalamic Neuron Resilience during Osmotic Demyelination Syndrome (ODS) Is Revealed by Primary Cilium Outgrowth and ADP-ribosylation factor-like protein 13B Labeling in Axon Initial Segment.

A murine osmotic demyelinating syndrome (ODS) model was developed through chronic hyponatremia, induced by desmopressin subcutaneous implants, followed by precipitous sodium restoration. The thalamic ventral posterolateral (VPL) and ventral posteromedial (VPM) relay nuclei were the most demyelinated regions where neuroglial damage could be evidenced without immune response. This report showed that following chronic hyponatremia, 12 h and 48 h time lapses after rebalancing osmolarity, amid the ODS-degraded outskirts, some resilient neuronal cell bodies built up primary cilium and axon hillock regions that extended into axon initial segments (AIS) where ADP-ribosylation factor-like protein 13B (ARL13B)-immunolabeled rod-like shape content was revealed. These AIS-labeled shaft lengths appeared proportional with the distance of neuronal cell bodies away from the ODS damaged epicenter and time lapses after correction of hyponatremia. Fine structure examination verified these neuron abundant transcriptions and translation regions marked by the ARL13B labeling associated with cell neurotubules and their complex cytoskeletal macromolecular architecture. This necessitated energetic transport to organize and restore those AIS away from the damaged ODS core demyelinated zone in the murine model. These labeled structures could substantiate how thalamic neuron resilience occurred as possible steps of a healing course out of ODS.

Aberrant axon initial segment plasticity and intrinsic excitability of ALS hiPSC motor neurons.

Dysregulated neuronal excitability is a hallmark of amyotrophic lateral sclerosis (ALS). We sought to investigate how functional changes to the axon initial segment (AIS), the site of action potential generation, could impact neuronal excitability in ALS human induced pluripotent stem cell (hiPSC) motor neurons. We find that early TDP-43 and C9orf72 hiPSC motor neurons show an increase in the length of the AIS and impaired activity-dependent AIS plasticity that is linked to abnormal homeostatic regulation of neuronal activity and intrinsic hyperexcitability. In turn, these hyperactive neurons drive increased spontaneous myofiber contractions of in vitro hiPSC motor units. In contrast, late hiPSC and postmortem ALS motor neurons show AIS shortening, and hiPSC motor neurons progress to hypoexcitability. At a molecular level, aberrant expression of the AIS master scaffolding protein ankyrin-G and AIS-specific voltage-gated sodium channels mirror these dynamic changes in AIS function and excitability. Our results point toward the AIS as an important site of dysfunction in ALS motor neurons.

Antibody-directed extracellular proximity biotinylation reveals that Contactin-1 regulates axo-axonic innervation of axon initial segments.

Axon initial segment (AIS) cell surface proteins mediate key biological processes in neurons including action potential initiation and axo-axonic synapse formation. However, few AIS cell surface proteins have been identified. Here, we use antibody-directed proximity biotinylation to define the cell surface proteins in close proximity to the AIS cell adhesion molecule Neurofascin. To determine the distributions of the identified proteins, we use CRISPR-mediated genome editing for insertion of epitope tags in the endogenous proteins. We identify Contactin-1 (Cntn1) as an AIS cell surface protein. Cntn1 is enriched at the AIS through interactions with Neurofascin and NrCAM. We further show that Cntn1 contributes to assembly of the AIS extracellular matrix, and regulates AIS axo-axonic innervation by inhibitory basket cells in the cerebellum and inhibitory chandelier cells in the cortex.

Temporal disparity of action potentials triggered in axon initial segments and distal axons in the neocortex.

Neural population activity determines the timing of synaptic inputs, which arrive to dendrites, cell bodies, and axon initial segments (AISs) of cortical neurons. Action potential initiation in the AIS (AIS-APs) is driven by input integration, and the phase preference of AIS-APs during network oscillations is characteristic to cell classes. Distal regions of cortical axons do not receive synaptic inputs, yet experimental induction protocols can trigger retroaxonal action potentials (RA-APs) in axons distal from the soma. We report spontaneously occurring RA-APs in human and rodent cortical interneurons that appear uncorrelated to inputs and population activity. Network-linked triggering of AIS-APs versus input-independent timing of RA-APs of the same interneurons results in disparate temporal contribution of a single cell to in vivo network operation through perisomatic and distal axonal firing.

Sodium channel endocytosis drives axon initial segment plasticity.

Activity-dependent plasticity of the axon initial segment (AIS) endows neurons with the ability to adapt action potential output to changes in network activity. Action potential initiation at the AIS highly depends on the clustering of voltage-gated sodium channels, but the molecular mechanisms regulating their plasticity remain largely unknown. Here, we developed genetic tools to label endogenous sodium channels and their scaffolding protein, to reveal their nanoscale organization and longitudinally image AIS plasticity in hippocampal neurons in slices and primary cultures. We find that N-methyl-d-aspartate receptor activation causes both long-term synaptic depression and rapid internalization of AIS sodium channels within minutes. The clathrin-mediated endocytosis of sodium channels at the distal AIS increases the threshold for action potential generation. These data reveal a fundamental mechanism for rapid activity-dependent AIS reorganization and suggests that plasticity of intrinsic excitability shares conserved features with synaptic plasticity.

Axon Initial Segment GABA Inhibits Action Potential Generation throughout Periadolescent Development.

Neurons are remarkably polarized structures: dendrites spread and branch to receive synaptic inputs while a single axon extends and transmits action potentials (APs) to downstream targets. Neuronal polarity is maintained by the axon initial segment (AIS), a region between the soma and axon proper that is also the site of action potential (AP) generation. This polarization between dendrites and axons extends to inhibitory neurotransmission. In adulthood, the neurotransmitter GABA hyperpolarizes dendrites but instead depolarizes axons. These differences in function collide at the AIS. Multiple studies have shown that GABAergic signaling in this region can share properties of either the mature axon or mature dendrite, and that these properties evolve over a protracted period encompassing periadolescent development. Here, we explored how developmental changes in GABAergic signaling affect AP initiation. We show that GABA at the axon initial segment inhibits action potential initiation in layer (L)2/3 pyramidal neurons in prefrontal cortex from mice of either sex across GABA reversal potentials observed in periadolescence. These actions occur largely through current shunts generated by GABAA receptors and changes in voltage-gated channel properties that affected the number of channels that could be recruited for AP electrogenesis. These results suggest that GABAergic neurons targeting the axon initial segment provide an inhibitory "veto" across the range of GABA polarity observed in normal adolescent development, regardless of GABAergic synapse reversal potential.Significance Statement GABA receptors are a major class of neurotransmitter receptors in the brain. Typically, GABA receptors inhibit neurons by allowing influx of negatively charged chloride ions into the cell. However, there are cases where local chloride concentrations promote chloride efflux through GABA receptors. Such conditions exist early in development in neocortical pyramidal cell axon initial segments (AISs), where action potentials (APs) initiate. Here, we examined how chloride efflux in early development interacts with mechanisms that support action potential initiation. We find that this efflux, despite moving membrane potential closer to action potential threshold, is nevertheless inhibitory. Thus, GABA at the axon initial segment is likely to be inhibitory for action potential initiation independent of whether chloride flows out or into neurons via these receptors.

Direct fluorescent labeling of NF186 and NaV1.6 in living primary neurons using bioorthogonal click chemistry.

The axon initial segment (AIS) is a highly specialized neuronal compartment that regulates the generation of action potentials and maintenance of neuronal polarity. Live imaging of the AIS is challenging due to the limited number of suitable labeling methods. To overcome this limitation, we established a novel approach for live labeling of the AIS using unnatural amino acids (UAAs) and click chemistry. The small size of UAAs and the possibility of introducing them virtually anywhere into target proteins make this method particularly suitable for labeling of complex and spatially restricted proteins. Using this approach, we labeled two large AIS components, the 186 kDa isoform of neurofascin (NF186; encoded by Nfasc) and the 260 kDa voltage-gated Na+ channel (NaV1.6, encoded by Scn8a) in primary neurons and performed conventional and super-resolution microscopy. We also studied the localization of epilepsy-causing NaV1.6 variants with a loss-of-function effect. Finally, to improve the efficiency of UAA incorporation, we developed adeno-associated viral (AAV) vectors for click labeling in neurons, an achievement that could be transferred to more complex systems such as organotypic slice cultures, organoids, and animal models.

ANK2 loss-of-function variants are associated with epilepsy, and lead to impaired axon initial segment plasticity and hyperactive network activity in hiPSC-derived neuronal networks.

PURPOSE: To characterize a novel neurodevelopmental syndrome due to loss-of-function (LoF) variants in Ankyrin 2 (ANK2), and to explore the effects on neuronal network dynamics and homeostatic plasticity in human-induced pluripotent stem cell-derived neurons. METHODS: We collected clinical and molecular data of 12 individuals with heterozygous de novo LoF variants in ANK2. We generated a heterozygous LoF allele of ANK2 using CRISPR/Cas9 in human-induced pluripotent stem cells (hiPSCs). HiPSCs were differentiated into excitatory neurons, and we measured their spontaneous electrophysiological responses using micro-electrode arrays (MEAs). We also characterized their somatodendritic morphology and axon initial segment (AIS) structure and plasticity. RESULTS: We found a broad neurodevelopmental disorder (NDD), comprising intellectual disability, autism spectrum disorders and early onset epilepsy. Using MEAs, we found that hiPSC-derived neurons with heterozygous LoF of ANK2 show a hyperactive and desynchronized neuronal network. ANK2-deficient neurons also showed increased somatodendritic structures and altered AIS structure of which its plasticity is impaired upon activity-dependent modulation. CONCLUSIONS: Phenotypic characterization of patients with de novo ANK2 LoF variants defines a novel NDD with early onset epilepsy. Our functional in vitro data of ANK2-deficient human neurons show a specific neuronal phenotype in which reduced ANKB expression leads to hyperactive and desynchronized neuronal network activity, increased somatodendritic complexity and AIS structure and impaired activity-dependent plasticity of the AIS.

Contribution of Axon Initial Segment Structure and Channels to Brain Pathology.

Brain channelopathies are a group of neurological disorders that result from genetic mutations affecting ion channels in the brain. Ion channels are specialized proteins that play a crucial role in the electrical activity of nerve cells by controlling the flow of ions such as sodium, potassium, and calcium. When these channels are not functioning properly, they can cause a wide range of neurological symptoms such as seizures, movement disorders, and cognitive impairment. In this context, the axon initial segment (AIS) is the site of action potential initiation in most neurons. This region is characterized by a high density of voltage-gated sodium channels (VGSCs), which are responsible for the rapid depolarization that occurs when the neuron is stimulated. The AIS is also enriched in other ion channels, such as potassium channels, that play a role in shaping the action potential waveform and determining the firing frequency of the neuron. In addition to ion channels, the AIS contains a complex cytoskeletal structure that helps to anchor the channels in place and regulate their function. Therefore, alterations in this complex structure of ion channels, scaffold proteins, and specialized cytoskeleton may also cause brain channelopathies not necessarily associated with ion channel mutations. This review will focus on how the AISs structure, plasticity, and composition alterations may generate changes in action potentials and neuronal dysfunction leading to brain diseases. AIS function alterations may be the consequence of voltage-gated ion channel mutations, but also may be due to ligand-activated channels and receptors and AIS structural and membrane proteins that support the function of voltage-gated ion channels.

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.

Nav1.2 and BK channel interaction shapes the action potential in the axon initial segment.

In neocortical layer-5 pyramidal neurons, the action potential (AP) is generated in the axon initial segment (AIS) when the membrane potential (Vm ) reaches the threshold for activation of the voltage-gated Na+ channels (VGNCs) Nav 1.2 and Nav 1.6. Yet, whereas these VGNCs are known to differ in spatial distribution along the AIS and in biophysical properties, our understanding of the functional differences between the two channels remains elusive. Here, using ultrafast Na+ , Vm and Ca2+ imaging in combination with partial block of Nav 1.2 by the peptide G1 G4 -huwentoxin-IV, we demonstrate an exclusive role of Nav 1.2 in shaping the generating AP. Precisely, we show that selective block of ∼30% of Nav 1.2 widens the AP in the distal part of the AIS and we demonstrate that this effect is due to a loss of activation of BK Ca2+ -activated K+ channels (CAKCs). Indeed, Ca2+ influx via Nav 1.2 activates BK CAKCs, determining the amplitude and the early phase of repolarization of the AP in the AIS. By using control experiments using 4,9-anhydrotetrodotoxin, a moderately selective inhibitor of Nav 1.6, we concluded that the Ca2+ influx shaping the early phase of the AP is exclusive of Nav 1.2. Hence, we mimicked this result with a neuron model in which the role of the different ion channels tested reproduced the experimental evidence. The exclusive role of Nav 1.2 reported here is important for understanding the physiology and pathology of neuronal excitability. KEY POINTS: We optically analysed the action potential generated in the axon initial segment of mouse layer-5 neocortical pyramidal neurons and its associated Na+ and Ca2+ currents using ultrafast imaging techniques. We found that partial selective block of the voltage-gated Na+ channel Nav 1.2, produced by a recently developed peptide, widens the shape of the action potential in the distal part of the axon initial segment. We demonstrate that this effect is due to a reduction of the Ca2+ influx through Nav 1.2 that activates BK Ca2+ -activated K+ channels. To validate our conclusions, we generated a neuron model that reproduces the ensemble of our experimental results. The present results indicate a specific role of Nav 1.2 in the axon initial segment for shaping of the action potential during its generation.

Carbogen-Induced Respiratory Acidosis Blocks Experimental Seizures by a Direct and Specific Inhibition of NaV1.2 Channels in the Axon Initial Segment of Pyramidal Neurons.

Brain pH is a critical factor for determining neuronal activity, with alkalosis increasing and acidosis reducing excitability. Acid shifts in brain pH through the breathing of carbogen (5% CO2/95% O2) reduces seizure susceptibility in animal models and patients. The molecular mechanisms underlying this seizure protection remain to be fully elucidated. Here, we demonstrate that male and female mice exposed to carbogen are fully protected from thermogenic-triggered seizures. Whole-cell patch-clamp recordings revealed that acid shifts in extracellular pH (pHo) significantly reduce action potential firing in CA1 pyramidal neurons but did not alter firing in hippocampal inhibitory interneurons. In real-time dynamic clamp experiments, acidification reduced simulated action potential firing generated in hybrid model neurons expressing the excitatory neuron predominant NaV1.2 channel. Conversely, acidification had no effect on action potential firing in hybrid model neurons expressing the interneuron predominant NaV1.1 channel. Furthermore, knockdown of Scn2a mRNA in vivo using antisense oligonucleotides reduced the protective effects of carbogen on seizure susceptibility. Both carbogen-mediated seizure protection and the reduction in CA1 pyramidal neuron action potential firing by low pHo were maintained in an Asic1a knock-out mouse ruling out this acid-sensing channel as the underlying molecular target. These data indicate that the acid-mediated reduction in excitatory neuron firing is mediated, at least in part, through the inhibition of NaV1.2 channels, whereas inhibitory neuron firing is unaffected. This reduction in pyramidal neuron excitability is the likely basis of seizure suppression caused by carbogen-mediated acidification.SIGNIFICANCE STATEMENT Brain pH has long been known to modulate neuronal excitability. Here, we confirm that brain acidification reduces seizure susceptibility in a mouse model of thermogenic seizures. Extracellular acidification reduced excitatory pyramidal neuron firing while having no effect on interneuron firing. Acidification also reduced dynamic clamp firing in cells expressing the NaV1.2 channel but not in cells expressing NaV1.1 channels. In vivo knockdown of Scn2a mRNA reduced seizure protection of acidification. In contrast, acid-mediated seizure protection was maintained in the Asic1a knock-out mouse. These data suggest NaV1.2 channel as an important target for acid-mediated seizure protection. Our results have implications on how natural variations in pH can modulate neuronal excitability and highlight potential antiseizure drug development strategies based on the NaV1.2 channel.