Voltage-gated sodium (Na) channels contribute to the regulation of cellular excitability due to their role in the generation and propagation of action potentials. They are composed of a pore-forming alpha subunit and are modulated by at least two of four distinct beta subunits (beta1-4). Recent studies have implicated a role for the intracellular domain of beta subunits in modulating Na channel gating and trafficking. In beta3, the intracellular domain contains a serine residue at position 161 that is replaced by an alanine in beta1. In this study, we have probed the functional importance of beta3S161 for modulating Na channel gating. Wild-type beta3 and point mutations beta3S161A or beta3S161E were individually co-expressed in HEK 293 cells stably expressing human Na(v)1.2. WTbeta3 expression increased Na current density, shifted steady-state inactivation in a depolarized direction, and accelerated the kinetics of recovery from inactivation of the Na current. Analogous effects were observed with beta3S161E co-expression. In contrast, beta3S161A abolished the shifts in steady-state inactivation and recovery from inactivation of the Na current, but did increase Na current density. Immunocytochemistry and Western blot experiments demonstrate membrane expression of WTbeta3, beta3S161E, and beta3S161A, suggesting that the differences in Na channel gating were not due to disruptions in beta subunit trafficking. These studies suggest that modification of beta3S161 may be important in modulating Na-channel gating.

Here we report on a novel fluorescent analog of phenytoin as a potential inhibitor of neuropathic pain with potential use as an imaging agent. Compound 2 incorporated a heptyl side chain and dansyl moiety onto the parent compound phenytoin and produced greater displacement of BTX from sodium channels and greater functional blockade with greatly reduced toxicity. Compound 2 reduced mechano-allodynia in a rat model of neuropathic pain and was visualized ex vivo in sensory neuron axons with two-photon microscopy. These results suggest a promising strategy for developing novel sodium channel inhibitors with imaging capabilities.

Human genetic studies show that the voltage gated sodium channel 1.7 (Nav1.7) is a key molecular determinant of pain sensation. However, defining the Nav1.7 contribution to nociceptive signalling has been hampered by a lack of selective inhibitors. Here we report two potent and selective arylsulfonamide Nav1.7 inhibitors; PF-05198007 and PF-05089771, which we have used to directly interrogate Nav1.7's role in nociceptor physiology. We report that Nav1.7 is the predominant functional TTX-sensitive Nav in mouse and human nociceptors and contributes to the initiation and the upstroke phase of the nociceptor action potential. Moreover, we confirm a role for Nav1.7 in influencing synaptic transmission in the dorsal horn of the spinal cord as well as peripheral neuropeptide release in the skin. These findings demonstrate multiple contributions of Nav1.7 to nociceptor signalling and shed new light on the relative functional contribution of this channel to peripheral and central noxious signal transmission.

Sudden unexpected death in epilepsy (SUDEP) accounts for the deaths of 8-17% of patients with epilepsy. Although the mechanisms of SUDEP are unknown, one proposed mechanism is abnormal control of the heart by the autonomic nervous system (ANS). Our objective was to determine whether the broad changes in ictal heart rate experienced by mouse models of SUDEP are (1) due to the ANS and (2) contribute to seizure-induced death. Seizures were induced by electrical stimulation of the hippocampus of a mouse carrying the human SCN8A encephalopathy mutation p.Asn1768Asp (N1768D; "D/+ mice"). Using standard autonomic pharmacology, the relative roles of the parasympathetic and sympathetic nervous systems on heart rate changes associated with seizures were determined. All induced seizures had pronounced ictal bradycardia and postictal tachycardia. Seizure susceptibility or severity were unchanged by the pharmacological agents. Administration of Atropine, a muscarinic antagonist, eliminated ictal bradycardia, while carbachol, a muscarinic agonist, had no effect on ictal bradycardia, but reduced postictal tachycardia. Sotalol, an adrenergic β-receptor antagonist, had no effect on ictal bradycardia, but did suppress postictal tachycardia. Isoproterenol, a β-receptor agonist, had no effect on either ictal bradycardia or postictal tachycardia. Administration of the α1-receptor antagonist prazosin increases the incidence of seizure-induced death in D/+ mice. Although postictal heart rate was lower for these fatal seizures in the presence of prazosin, rates were not as low as that recorded for carbachol treated mice, which all survived. Both ictal bradycardia and postictal tachycardia are manifestations of the ANS. Bradycardia is mediated by a maximal activation of the parasympathetic arm of the ANS, and tachycardia is mediated by parasympathetic inactivation and sympathetic activation. While the changes in heart rate during seizures are profound, suppression of postictal heart rate did not increase seizure mortality.

SCN8A encephalopathy, or early infantile epileptic encephalopathy 13 (EIEE13), is caused predominantly by de novo gain-of-function mutations in the voltage-gated Na channel Nav1.6. Affected individuals suffer from refractory seizures, developmental delay, cognitive disability, and elevated risk of sudden unexpected death in epilepsy (SUDEP). A knock-in mouse model carrying the patient mutation p.Asn1768Asp (N1768D) reproduces many features of the disorder, including spontaneous seizures and SUDEP. We used the mouse model to examine the effects of the mutation on layer II stellate neurons of the medial entorhinal cortex (mEC), which transmit excitatory input to the hippocampus. Heterozygous (Scn8aD/+), homozygous (Scn8aD/D)), and WT (Scn8a+/+) littermates were compared at 3 weeks of age, the time of seizure onset for homozygous mice. Heterozygotes remain seizure free for another month. mEC layer II neurons of heterozygous and homozygous mice were hyperexcitable and generated long-lasting depolarizing potentials with bursts of action potentials after synaptic stimulation. Recording of Na currents revealed proexcitatory increases in persistent and resurgent currents and rightward shifts in inactivation parameters, leading to significant increases in the magnitude of window currents. The proexcitatory changes were more pronounced in homozygous mice than in heterozygotes, consistent with the earlier age of seizure onset in homozygotes. These studies demonstrate that the N1768D mutation increases the excitability of mEC layer II neurons by increasing persistent and resurgent Na currents and disrupting channel inactivation. The aberrant activities of mEC layer II neurons would provide excessive excitatory input to the hippocampus and contribute to hyperexcitability of hippocampal neurons in this model of SCN8A encephalopathy.SIGNIFICANCE STATEMENTSCN8A encephalopathy is a devastating neurological disorder that results from de novo mutations in the Na channel Nav1.6. In addition to seizures, patients suffer from cognitive and developmental delays and are at high risk for sudden unexpected death in epilepsy (SUDEP). A mouse knock-in model expressing the patient mutation N1768D reproduces several pathological phenotypes, including spontaneous seizures and sudden death. We demonstrate that medial entorhinal cortex (mEC) neurons from the mouse model exhibit proexcitatory alterations in Na channel activity, some of which were not seen in hippocampal or cortical neurons, and resulting in neuronal hyperexcitability. Because mEC neurons regulate the activity of the hippocampus, which plays an important role in seizure onset, we propose that these profound changes in mEC neuron excitability associated with the gain-of-function mutation of Nav1.6 may increase excitatory drive into the hippocampus, culminating in seizure activity and SUDEP.

Dravet Syndrome (DS) is a severe developmental and epileptic encephalopathy typically caused by loss-of-function de novo mutations in the SCN1A gene which encodes the voltage-gated sodium channel isoform NaV1.1. Decreased NaV1.1 expression results in impaired excitability of inhibitory interneurons and seizure onset. To date, there are no clinically available treatments for DS that directly address the core mechanism of disease; reduced NaV1.1 expression levels in interneurons. Recently, Targeted Augmentation of Nuclear Gene Output (TANGO) of SCN1A by the antisense oligonucleotide (ASO) STK-001, was shown to increase Scn1a mRNA levels, increase NaV1.1 protein expression, reduce seizures, and improve survival in the Scn1a+/- mouse model of DS. However, it remains unknown whether STK-001 treatment rescues the reduced intrinsic excitability of parvalbumin-positive (PV) inhibitory interneurons associated with DS. In this study, we demonstrate that STK-001 treatment reduces seizures, prolongs survival, and rescues PV interneuron excitability in Scn1a+/- mice to levels observed in WT littermates. Together, these results support the notion that TANGO-mediated augmentation of NaV1.1 levels directly targets and rescues one of the core disease mechanisms of DS.

In this work, carbon dots (CDs) were synthesized by a one-step hydrothermal method using citric acid and ethylene diamine, and covalently functionalized with antibodies for the sensing of progesterone hormone. The structural and morphological analysis reveals that the synthesized CDs are of average size (diameter 8-10 nm) and the surface functionalities are confirmed by XPS, XRD and FT-IR. Further graphene oxide (GO) is used as a quencher due to the fluorescence resonance energy transfer (FRET) mechanism, whereas the presence of the analyte progesterone turns on the fluorescence because of displacement of GO from the surface of CDs effectively inhibiting FRET efficiency due to the increased distance between donor and acceptor moieties. The linear curve is obtained with different progesterone concentrations with 13.8 nM detection limits (R2 = 0.974). The proposed optical method demonstrated high selectivity performance in the presence of structurally resembling interfering compounds. The PL intensity increased linearly with the increased progesterone concentration range (10-900 nM) under the optimal experimental parameters. The developed level-free immunosensor has emerged as a potential platform for simplified progesterone analysis due to the high selectivity performance and good recovery in different samples of spiked water.

Temporal lobe epilepsy (TLE) is the most common form of adult epilepsy involving the limbic structures of the temporal lobe. Layer II neurons of the entorhinal cortex (EC) form the major excitatory input into the hippocampus via the perforant path and consist of non-stellate and stellate neurons. These neurons are spared and hyper-excitable in TLE. The basis for the hyper-excitability is likely multifactorial and may include alterations in intrinsic properties. In a rat model of TLE, medial EC (mEC) non-stellate and stellate neurons had significantly higher action potential (AP) firing frequencies than in control. The increase remained in the presence of synaptic blockers, suggesting intrinsic mechanisms. Since sodium (Na) channels play a critical role in AP generation and conduction we sought to determine if Na channel gating parameters and expression levels were altered in TLE. Na channel currents recorded from isolated mEC TLE neurons revealed increased Na channel conductances, depolarizing shifts in inactivation parameters and larger persistent (I(NaP)) and resurgent (I(NaR)) Na currents. Immunofluorescence experiments revealed increased staining of Na(v)1.6 within the axon initial segment and Na(v)1.2 within the cell bodies of mEC TLE neurons. These studies provide support for additional intrinsic alterations within mEC layer II neurons in TLE and implicate alterations in Na channel activity and expression, in part, for establishing the profound increase in intrinsic membrane excitability of mEC layer II neurons in TLE. These intrinsic changes, together with changes in the synaptic network, could support seizure activity in TLE.

Optogenetic and chemogenetic actuators are critical for deconstructing the neural correlates of behavior. However, these tools have several limitations, including invasive modes of stimulation or slow on/off kinetics. We have overcome these disadvantages by synthesizing a single-component, magnetically sensitive actuator, "Magneto," comprising the cation channel TRPV4 fused to the paramagnetic protein ferritin. We validated noninvasive magnetic control over neuronal activity by demonstrating remote stimulation of cells using in vitro calcium imaging assays, electrophysiological recordings in brain slices, in vivo electrophysiological recordings in the brains of freely moving mice, and behavioral outputs in zebrafish and mice. As proof of concept, we used Magneto to delineate a causal role of striatal dopamine receptor 1 neurons in mediating reward behavior in mice. Together our results present Magneto as an actuator capable of remotely controlling circuits associated with complex animal behaviors.

Although T-type Ca(2+) channels in the thalamus play a crucial role in determining neuronal excitability and are involved in sensory processing and pathophysiology of epilepsy, little is known about the molecular mechanisms involved in their regulation. Here, we report that reducing agents, including endogenous sulfur-containing amino acid l-cysteine, selectively enhance native T-type currents in reticular thalamic (nRT) neurons and recombinant Ca(V)3.2 (alpha1H) currents, but not native and recombinant Ca(V)3.1 (alpha1G)- and Ca(V)3.3 (alpha1I)-based currents. Consistent with this data, T-type currents of nRT neurons from transgenic mice lacking Ca(V)3.2 channel expression were not modulated by reducing agents. In contrast, oxidizing agents inhibited all native and recombinant T-type currents non-selectively. Thus, our findings directly demonstrate that Ca(V)3.2 channels are the main molecular substrate for redox regulation of neuronal T-type channels. In addition, because thalamic T-type channels generate low-threshold Ca(2+) spikes that directly correlate with burst firing in these neurons, differential redox regulation of these channels may have an important function in controlling cellular excitability in physiological and pathological conditions and fine-tuning of the flow of sensory information into the central nervous system.

The medial prefrontal cortex (mPFC) is involved in a wide range of executive cognitive functions, including reward evaluation, decision-making, memory extinction, mood, and task switching. Manipulation of the mPFC has been shown to alter food intake and food reward valuation, but whether exclusive stimulation of mPFC pyramidal neurons (PN), which form the principle output of the mPFC, is sufficient to mediate food rewarded instrumental behavior is unknown. We sought to determine the behavioral consequences of manipulating mPFC output by exciting PN in mouse mPFC during performance of a panel of behavioral assays, focusing on food reward. We found that increasing mPFC pyramidal cell output using designer receptors exclusively activated by designer drugs (DREADD) enhanced performance in instrumental food reward assays that assess food seeking behavior, while sparing effects on affect and food intake. Specifically, activation of mPFC PN enhanced operant responding for food reward, reinstatement of palatable food seeking, and suppression of impulsive responding for food reward. Conversely, activation of mPFC PN had no effect on unconditioned food intake, social interaction, or behavior in an open field. Furthermore, we found that behavioral outcome is influenced by the degree of mPFC activation, with a low drive sufficient to enhance operant responding and a higher drive required to alter impulsivity. Additionally, we provide data demonstrating that DREADD stimulation involves a nitric oxide (NO) synthase dependent pathway, similar to endogenous muscarinic M3 receptor stimulation, a finding that provides novel mechanistic insight into an increasingly widespread method of remote neuronal control.

Sodium (Na) channel blockers are known to possess antihyperalgesic properties. We have designed and synthesized a novel Na channel antagonist, alpha-hydroxyphenylamide, and determined its ability to inhibit both TTX-sensitive (TTX-s) and TTX-resistant (TTX-r) Na currents from small dorsal root ganglion (DRG) neurons. alpha-Hydroxyphenylamide tonically inhibited both TTX-s and TTX-r Na currents yielding an IC(50) of 8.2+/-2.2 microM (n=7) and 28.9+/-1.8 microM (n=8), respectively. In comparison, phenytoin was less potent inhibiting TTX-s and TTX-r currents by 26.2+/-4.0% (n=8) and 25.5+/-2.0%, respectively, at 100 microM. alpha-Hydroxyphenylamide (10 microM) also shifted equilibrium gating parameters of TTX-s Na channels to greater hyperpolarized potentials, slowed recovery from inactivation, accelerated the development of inactivation and exhibited use-dependent block. In the chronic constriction injury (CCI) rat model of neuropathic pain, intraperitoneal administration of alpha-hydroxyphenylamide attenuated the hyperalgesia by 53% at 100mg/kg, without affecting motor coordination in the Rotorod test. By contrast, the reduction in pain behavior produced by phenytoin (73%; 100mg/kg) was associated with significant motor impairment. In summary, we report that alpha-hydroxyphenylamide, a sodium channel antagonist, exhibits antihyperalgesic properties in a rat model of neuropathic pain, with favorable sedative and ataxic side effects compared with phenytoin.

The putative cache (Ca2+ channel and chemotaxis receptor) domain containing 1 (CACHD1) protein has predicted structural similarities to members of the α2δ voltage-gated Ca2+ channel auxiliary subunit family. CACHD1 mRNA and protein were highly expressed in the male mammalian CNS, in particular in the thalamus, hippocampus, and cerebellum, with a broadly similar tissue distribution to CaV3 subunits, in particular CaV3.1. In expression studies, CACHD1 increased cell-surface localization of CaV3.1, and these proteins were in close proximity at the cell surface, consistent with the formation of CACHD1-CaV3.1 complexes. In functional electrophysiological studies, coexpression of human CACHD1 with CaV3.1, CaV3.2, and CaV3.3 caused a significant increase in peak current density and corresponding increases in maximal conductance. By contrast, α2δ-1 had no effect on peak current density or maximal conductance in CaV3.1, CaV3.2, or CaV3.3. A comparison of CACHD1-mediated increases in CaV3.1 current density and gating currents revealed an increase in channel open probability. In hippocampal neurons from male and female embryonic day 19 rats, CACHD1 overexpression increased CaV3-mediated action potential firing frequency and neuronal excitability. These data suggest that CACHD1 is structurally an α2δ-like protein that functionally modulates CaV3 voltage-gated calcium channel activity.SIGNIFICANCE STATEMENT This is the first study to characterize the Ca2+ channel and chemotaxis receptor domain containing 1 (CACHD1) protein. CACHD1 is widely expressed in the CNS, in particular in the thalamus, hippocampus, and cerebellum. CACHD1 distribution is similar to that of low voltage-activated (CaV3, T-type) calcium channels, in particular to CaV3.1, a protein that regulates neuronal excitability and is a potential therapeutic target in conditions such as epilepsy and pain. CACHD1 is structurally an α2δ-like protein that functionally increases CaV3 calcium current. CACHD1 increases the presence of CaV3.1 at the cell surface, forms complexes with CaV3.1 at the cell surface, and causes an increase in channel open probability. In hippocampal neurons, CACHD1 causes increases in neuronal firing. Thus, CACHD1 represents a novel protein that modulates CaV3 activity.

Epileptogenesis is characterized by intrinsic changes in neuronal firing, resulting in hyperactive neurons and the subsequent generation of seizure activity. These alterations are accompanied by changes in gene transcription networks, first with the activation of early-immediate genes and later with the long-term activation of genes involved in memory. Our objective was to engineer a promoter containing binding sites for activity-dependent transcription factors upregulated in chronic epilepsy (EpiPro) and validate it in multiple rodent models of epilepsy. First, we assessed the activity dependence of EpiPro: initial electrophysiology studies found that EpiPro-driven GFP expression was associated with increased firing rates when compared with unlabeled neurons, and the assessment of EpiPro-driven GFP expression revealed that GFP expression was increased ~150× after status epilepticus. Following this, we compared EpiPro-driven GFP expression in two rodent models of epilepsy, rat lithium/pilocarpine and mouse electrical kindling. In rodents with chronic epilepsy, GFP expression was increased in most neurons, but particularly in dentate granule cells, providing in vivo evidence to support the "breakdown of the dentate gate" hypothesis of limbic epileptogenesis. Finally, we assessed the time course of EpiPro activation and found that it was rapidly induced after seizures, with inactivation following over weeks, confirming EpiPro's potential utility as a gene therapy driver for epilepsy.

The early infantile epileptic encephalopathy type 13 (EIEE13, OMIM #614558) results from de novo missense mutations of SCN8A encoding the voltage-gated sodium channel Nav1.6. More than 20% of patients have recurrent mutations in residues Arg1617 or Arg1872. Our goal was to determine the functional effects of these mutations on channel properties.

Temporal lobe epilepsy (TLE) is a form of adult epilepsy involving the entorhinal cortex (EC). Layer II neurons of the medial EC (mEC) are spared and become hyperexcitable in TLE. Studies have suggested a role for T-type calcium channels (T-type Ca2+ channels) in facilitating increases in neuronal activity associated with TLE within the hippocampus. We sought to determine if T-type Ca2+ channels play a role in facilitating neuronal hyperexcitability of layer II mEC stellate neurons in TLE. TLE was induced in rats by electrical stimulation of the hippocampus to induce status epilepticus (SE). Brain slices were prepared from rats exhibiting spontaneous seizures and compared with age-matched control rats. Action potentials (APs) were evoked either by current injection steps or via presynaptic stimulation of mEC deep layers. The selective T-type Ca2+ channel antagonist, TTA-P2 (1 μM), was applied to determine the role of T-type Ca2+ channels in maintaining neuronal excitability. Quantitative PCR techniques were used to assess T-type Ca2+ channel isoform mRNA levels within the mEC layer II. TLE mEC layer II stellate neurons were hyperexcitable compared to control neurons, evoking a higher frequency of APs and generating bursts of APs when synaptically stimulated. TTA-P2 (1 μM) reduced firing frequencies in TLE and control neurons and reduced AP burst firing in TLE stellate neurons. TTA-P2 had little effect on synaptically evoked AP's in control neurons. TTA-P2 also inhibited rebound APs evoked in TLE neurons to a greater degree than in control neurons. TLE tissue had almost a 3-fold increase in Cav3.1 mRNA compared to controls. Cav3.2 or Cav3.3 levels were unchanged. These findings support a role for T-type Ca2+ channel in establishing neuronal hyperexcitability of mEC layer II stellate neurons in TLE. Increased expression of Cav3.1 may be important for establishing neuronal hyperexcitability of mEC layer II neurons in TLE.

SCN8A epileptic encephalopathy is caused predominantly by de novo gain-of-function mutations in the voltage-gated sodium channel Nav 1.6. The disorder is characterized by early onset of seizures and developmental delay. Most patients with SCN8A epileptic encephalopathy are refractory to current anti-seizure medications. Previous studies determining the mechanisms of this disease have focused on neuronal dysfunction as Nav 1.6 is expressed by neurons and plays a critical role in controlling neuronal excitability. However, glial dysfunction has been implicated in epilepsy and alterations in glial physiology could contribute to the pathology of SCN8A encephalopathy. In the current study, we examined alterations in astrocyte and microglia physiology in the development of seizures in a mouse model of SCN8A epileptic encephalopathy.