Voltage-gated Sodium Channel Currents Research Articles

Low-frequency rTMS modulated the excitability and high-frequency firing in hippocampal neurons of the Alzheimer’s disease mouse model

Repetitive transcranial magnetic stimulation (rTMS), a non-invasive brain stimulation technique, holds potential for applications in the treatment of Alzheimer’s disease (AD). This study aims to compare the therapeutic effects of rTMS at different frequencies on Alzheimer’s disease and explore the alterations in neuronal electrophysiological properties throughout this process. APP/PS1 AD mice were subjected to two rTMS treatments at 0.5 Hz and 20 Hz, followed by assessments of therapeutic outcomes through the Novel Object Recognition (NOR) and Morris Water Maze (MWM) tests. Following this, whole-cell patch-clamp techniques were used to record action potential, voltage-gated sodium channel currents, and voltage-gated potassium channel currents in dentate gyrus granule neurons. The results show that AD mice exhibit significant cognitive decline compared to normal mice, along with a pronounced reduction in neuronal excitability and ion channel activity. Both frequencies of rTMS treatment partially reversed these changes, demonstrating similar therapeutic efficacy. Furthermore, the investigation indicates that low-frequency magnetic stimulation inhibited the concentrated firing of early action potentials in AD.

High calcium concentrations reduce cellular excitability of mouse MNTB neurons

Calcium, a universal intracellular signaling molecule, plays essential roles in neural functions. Historically, in most in vitro brain slice electrophysiology studies, the extracellular calcium concentration ([Ca2+]e) in artificial cerebrospinal fluid is of a wide range and typically higher than the physiological value. At high [Ca2+]e, synaptic transmission is generally enhanced. However, the effects and the underlying mechanisms of calcium on intrinsic neuronal properties are diverse. Using whole-cell patch clamp in acute brainstem slices obtained from mice of either sex, we investigated the effects and the underlying mechanisms of high [Ca2+]e on intrinsic neuronal properties of neurons in the medial nucleus of the trapezoid body (MNTB), an auditory brainstem component in the sound localization circuitry. Compared to the physiological [Ca2+]e (1.2 mM), high [Ca2+]e at 1.8 and 2.4 mM significantly reduced the cellular excitability of MNTB neurons, resulting in decreased spike firing rate, depolarized spike threshold, and decreased the ability to follow high frequency inputs. High extracellular magnesium concentrations at 1.8 and 2.4 mM produced similar but less robust effects, due to surface charge screening. Upon high calcium application, voltage-gated sodium channel currents remained largely unchanged. Calcium-sensing receptors were detected in MNTB neurons, but blocking these receptors did not eliminate the effects of high calcium on spontaneous spiking. We attribute the lack of significant effects in these last two experiments to the moderate changes in calcium we tested. Our results call for the use of physiological [Ca2+]e in brain slice experiments.

The volatile anesthetic isoflurane differentially inhibits voltage-gated sodium channel currents between pyramidal and parvalbumin neurons in the prefrontal cortex.

How volatile anesthetics work remains poorly understood. Modulations of synaptic neurotransmission are the direct cellular mechanisms of volatile anesthetics in the central nervous system. Volatile anesthetics such as isoflurane may reduce neuronal interaction by differentially inhibiting neurotransmission between GABAergic and glutamatergic synapses. Presynaptic voltage-dependent sodium channels (Nav), which are strictly coupled with synaptic vesicle exocytosis, are inhibited by volatile anesthetics and may contribute to the selectivity of isoflurane between GABAergic and glutamatergic synapses. However, it is still unknown how isoflurane at clinical concentrations differentially modulates Nav currents between excitatory and inhibitory neurons at the tissue level. In this study, an electrophysiological recording was applied in cortex slices to investigate the effects of isoflurane on Nav between parvalbumin (PV+) and pyramidal neurons in PV-cre-tdTomato and/or vglut2-cre-tdTomato mice. Isoflurane at clinically relevant concentrations produced a hyperpolarizing shift in the voltage-dependent inactivation and slowed the recovery time from the fast inactivation in both cellular subtypes. Since the voltage of half-maximal inactivation was significantly depolarized in PV+ neurons compared to that of pyramidal neurons, isoflurane inhibited the peak Nav currents in pyramidal neurons more potently than those of PV+ neurons (35.95 ± 13.32% vs. 19.24 ± 16.04%, P = 0.036 by the Mann-Whitney test). Isoflurane differentially inhibits Nav currents between pyramidal and PV+ neurons in the prefrontal cortex, which may contribute to the preferential suppression of glutamate release over GABA release, resulting in the net depression of excitatory-inhibitory circuits in the prefrontal cortex.

Inhibitory effect of atomoxetine on Nav1.2 voltage-gated sodium channel currents.

Atomoxetine (ATX), a norepinephrine reuptake inhibitor (NRI), is used to attenuate the symptoms of Attention Deficit/Hyperactivity Disorder (AD/HD) by increasing neurotransmitter concentrations at the synaptic cleft. Although Nav1.2 voltage-gated sodium channels (VGSCs) are thought to play a role in monoamine transmitter release in the synaptic junction, it is unclear how atomoxetine affects Nav1.2 VGSCs. In this study, we investigated the effect of ATX on Nav1.2 VGSC-transfected HEK293 cells with the whole-patch clamp technique. Nav1.2 VGSC current decreased by 51.15 ± 12.75% under treatment with 50µM ATX in the resting state (holding membrane potential at - 80mV). The IC50 of ATX against Nav1.2 VGSC current was 45.57µM. The activation/inactivation curve of Nav1.2 VGSC currents was shifted toward hyperpolarization by 50µM ATX. In addition, the inhibitory effect of ATX increased with membrane depolarization (holding membrane potential at - 50mV) and its IC50 was 10.16µM. Moreover, ATX showed the time-dependent interaction in the inactivation state. These findings suggest that ATX interacts with Nav1.2 VGSCs producing the inhibition of current and the modification of kinetic properties in the state-dependent manner.

Somatic and terminal CB1 receptors are differentially coupled to voltage-gated sodium channels in neocortical neurons

Endogenous cannabinoid signaling is vital for important brain functions, and the same pathways can be modified pharmacologically to treat pain, epilepsy, and posttraumatic stress disorder. Endocannabinoid-mediated changes to excitability are predominantly attributed to 2-arachidonoylglycerol (2-AG) acting presynaptically via the canonical cannabinoid receptor, CB1. Here, we identify a mechanism in the neocortex by which anandamide (AEA), another major endocannabinoid, but not 2-AG, powerfully inhibits somatically recorded voltage-gated sodium channel (VGSC) currents in the majority of neurons. This pathway involves intracellular CB1 that, when activated by anandamide, decreases the likelihood of recurrent action potential generation. WIN 55,212-2 similarly activates CB1 and inhibits VGSC currents, indicating that this pathway is also positioned to mediate the actions of exogenous cannabinoids on neuronal excitability. The coupling between CB1 and VGSCs is absent at nerve terminals, and 2-AG does not block somatic VGSC currents, indicating functional compartmentalization of the actions of two endocannabinoids.

Cinacalcet inhibition of neuronal action potentials preferentially targets the fast inactivated state of voltage-gated sodium channels.

Voltage-gated sodium channel (VGSC) activation is essential for action potential generation in the brain. Allosteric calcium-sensing receptor (CaSR) agonist, cinacalcet, strongly and ubiquitously inhibits VGSC currents in neocortical neurons via an unidentified, G-protein-dependent inhibitory molecule. Here, using whole-cell patch VGSC clamp methods, we investigated the voltage-dependence of cinacalcet-mediated inhibition of VGSCs and the channel state preference of cinacalcet. The rate of inhibition of VGSC currents was accelerated at more depolarized holding potentials. Cinacalcet shifted the voltage-dependence of both fast and slow inactivation of VGSC currents in the hyperpolarizing direction. Utilizing a simple model, the voltage-dependence of VGSC current inhibition may be explained if the affinity of the inhibitory molecule to the channel states follows the sequence: fast-inactivated > slow-inactivated > resting. The state dependence of VGSC current inhibition contributes to the non-linearity of action potential block by cinacalcet. This dynamic and abundant signaling pathway by which cinacalcet regulates VGSC currents provides an important voltage-dependent mechanism for modulating central neuronal excitability.

Critical Appraisal of Cenobamate as Adjunctive Treatment of Focal Seizures in Adults.

Cenobamate (CNB) is the latest antiseizure medication (ASM) authorized for the treatment of focal-onset seizures in adults. Although the precise mechanism of action of CNB is not yet fully understood, this drug inhibits the persistent, rather than transient, voltage-gated sodium channel currents and is a positive allosteric modulator of synaptic and extrasynaptic GABAA receptors, differently from benzodiazepines. CNB has a non-linear pharmacokinetic with a terminal half-life range of about 50/60 hours within the therapeutic dose range, which allows once daily administration. Cenobamate inhibits cytochrome P450 (CYP) 2C19 and induces CYP3A4 and 2B6, and hence can potentially interact with ASMs (eg, phenytoin, carbamazepine and clobazam) and no-ASMs drugs. In two randomized, double-blind, placebo-controlled trials in patients with focal epilepsies, CNB has shown a particularly good efficacy with a rate of seizure freedom of about 20% during the maintenance period in participants treated with the dose of 400 mg/day. The most common treatment-emergent adverse effects include central nervous system-related symptoms, like dizziness, diplopia, somnolence, and gait disturbances. Safety issues of particular interest are severe skin reactions (drug reaction with eosinophilia and systemic symptoms) and QT shortening, which contraindicates its use in subjects with familial short QT syndrome or in combination with other QT-shortening drugs. The recommended starting dose is 12.5 mg/day, which can be gradually titrated to the target dose (200 mg/day) and further increased up to 400 mg/day. There are several aspects of CNB that need to be still addressed, including the long-term efficacy and the efficacy in patients with generalized seizures. Ongoing studies will clarify these issues. The clinical relevance of the peculiar pharmacokinetics and the pattern of drug–drug interactions also require further investigation.

Neurotoxic and convulsant effects induced by jack bean ureases on the mammalian nervous system

Ureases are microbial virulence factors either because of the enzymatic release of ammonia or due to many other non-enzymatic effects. Here we studied two neurotoxic urease isoforms, Canatoxin (CNTX) and Jack Bean Urease (JBU), produced by the plant Canavalia ensiformis, whose mechanisms of action remain elusive. The neurotoxins provoke convulsions in rodents (LD50 ∼2 mg/kg) and stimulate exocytosis in cell models, affecting intracellular calcium levels. Here, electrophysiological and brain imaging techniques were applied to elucidate their mode of action. While systemic administration of the toxins causes tonic-clonic seizures in rodents, JBU injected into rat hippocampus induced spike-wave discharges similar to absence-like seizures. JBU reduced the amplitude of compound action potential from mouse sciatic nerve in a tetrodotoxin-insensitive manner. Hippocampal slices from CNTX-injected animals or slices treated in vitro with JBU failed to induce long term potentiation upon tetanic stimulation. Rat cortical synaptosomes treated with JBU released L-glutamate. JBU increased the intracellular calcium levels and spontaneous firing rate in rat hippocampus neurons. MicroPET scans of CNTX-injected rats revealed increased [18]Fluoro-deoxyglucose uptake in epileptogenesis-related areas like hippocampus and thalamus. Curiously, CNTX did not affect voltage-gated sodium, calcium or potassium channels currents, neither did it interfere on cholinergic receptors, suggesting an indirect mode of action that could be related to the ureases’ membrane-disturbing properties. Understanding the neurotoxic mode of action of C. ensiformis ureases could help to unveil the so far underappreciated relevance of these toxins in diseases caused by urease-producing microorganisms, in which the human central nervous system is affected.

Methylene blue induces an analgesic effect by significantly decreasing neural firing rates and improves pain behaviors in rats

Methylene blue (MB) is a blue cationic thiazine dye and currently used in different medical settings. Notably, there have been several attempts to introduce MB for attenuating pain in the last decade. Some clinical studies reported remarkable results, which, however, have been much debated. In addition, accumulating evidence have revealed that MB diminishes voltage-gated sodium channel currents. Accordingly, in the present study, we conducted in vivo experiments, including in vivo single nerve recording and behavioral test, to investigate whether MB dampens neural firing rates and ultimately contributes to pain relief. As a result, neural firing rates significantly decreased and finally converged to zero after MB administration. This event lasted longer than that of lidocaine and was dose-dependently modulated. Furthermore, there was a marked improvement in pain behaviors. The withdrawal threshold and latency of hind paws significantly rose post-MB administration. Therefore, these results demonstrate that MB lessens pain by significantly weakening neural excitability, which implies a strong possibility that this dye may be developed as a pain-relieving medication in the future. This is the first in vivo study to elucidate the effect of MB on nerves and pain relief.

Magnolol inhibits sodium currents in freshly isolated mouse dorsal root ganglion neurons.

The voltage-gated sodium channel (VGSC) currents in dorsal root ganglion (DRG) neurons contain mainly TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) Na+ currents. Magnolol (Mag), a hydroxylated biphenyl compound isolated from the bark of Magnolia officinalis, has been well documented to exhibit analgesic effects, but its mechanism is not yet fully understood. The aim of the present study was to investigate whether the antinociceptive effects of Mag is through inhibition of Na+ currents. Na+ currents in freshly isolated mouse DRG neurons were recorded with the whole cell patch clamp technique. Results showed that Mag inhibited TTX-S and TTX-R Na+ currents in a concentration-dependent manner. The IC50 values for block of TTX-S and TTX-R Na+ currents were 9.4 and 7.0μmol/L, respectively. Therefore, TTX-R Na+ current was more susceptible to Mag than TTX-S Na+ current. For TTX-S Na+ channel, 10μmol/L Mag shifted the steady state inactivation curve toward more negative by 9.8mV, without affecting the activation curve. For TTX-R Na+ channel, 7μmol/L Mag shifted the steady state activation and inactivation curves toward more positive and negative potentials by 6.5 and 11.7mV, respectively. In addition, Mag significantly postponed recovery of TTX-S and TTX-R Na+ currents from inactivation, and produced frequency dependent blocks of both subtypes of Na+ currents. These results suggest that the inhibitory effects of Mag on Na+ channels may contribute to its analgesic effect.

Jedi-1 deficiency increases sensory neuron excitability through a non-cell autonomous mechanism

The dorsal root ganglia (DRG) house the primary afferent neurons responsible for somatosensation, including pain. We previously identified Jedi-1 (PEAR1/MEGF12) as a phagocytic receptor expressed by satellite glia in the DRG involved in clearing apoptotic neurons during development. Here, we further investigated the function of this receptor in vivo using Jedi-1 null mice. In addition to satellite glia, we found Jedi-1 expression in perineurial glia and endothelial cells, but not in sensory neurons. We did not detect any morphological or functional changes in the glial cells or vasculature of Jedi-1 knockout mice. Surprisingly, we did observe changes in DRG neuron activity. In neurons from Jedi-1 knockout (KO) mice, there was an increase in the fraction of capsaicin-sensitive cells relative to wild type (WT) controls. Patch-clamp electrophysiology revealed an increase in excitability, with a shift from phasic to tonic action potential firing patterns in KO neurons. We also found alterations in the properties of voltage-gated sodium channel currents in Jedi-1 null neurons. These results provide new insight into the expression pattern of Jedi-1 in the peripheral nervous system and indicate that loss of Jedi-1 alters DRG neuron activity indirectly through an intercellular interaction between non-neuronal cells and sensory neurons.

The General Anesthetic Isoflurane Bilaterally Modulates Neuronal Excitability

SummaryVolatile anesthetics induce hyperactivity during induction while producing anesthesia at higher concentrations. They also bidirectionally modulate many neuronal functions. However, the neuronal mechanism is unclear. The effects of isoflurane on sodium channel currents were analyzed in acute mouse brain slices, including sodium leak (NALCN) currents and voltage-gated sodium channels (Nav) currents. Isoflurane at sub-anesthetic concentrations increased the spontaneous firing rate of CA3 pyramidal neurons, whereas anesthetic concentrations of isoflurane decreased the firing rate. Isoflurane at sub-anesthetic concentrations enhanced NALCN conductance but minimally inhibited Nav currents. Isoflurane at anesthetic concentrations depressed Nav currents and action potential amplitudes. Isoflurane at sub-anesthetic concentrations depolarized resting membrane potential (RMP) of neurons, whereas hyperpolarized the RMP at anesthetic concentrations. Isoflurane at low concentrations induced hyperactivity in vivo, which was diminished in NALCN knockdown mice. In conclusion, enhancement of NALCN by isoflurane contributes to its bidirectional modulation of neuronal excitability and the hyperactivity during induction.

Ca2+-dependent recruitment of voltage-gated sodium channels underlies bilirubin-induced overexcitation and neurotoxicity

Neonatal jaundice is prevalent among newborns and can lead to severe neurological deficits, particularly sensorimotor dysfunction. Previous studies have shown that bilirubin (BIL) enhances the intrinsic excitability of central neurons and this can potentially contribute to their overexcitation, Ca2+ overload, and neurotoxicity. However, the cellular mechanisms underlying elevated neuronal excitability remain unknown. By performing patch-clamp recordings from neonatal neurons in the rat medial vestibular nucleus (MVN), a crucial relay station for locomotor and balance control, we found that BIL (3 μM) drastically increases the spontaneous firing rates by upregulating the current-mediated voltage-gated sodium channels (VGSCs), while shifting their voltage-dependent activation toward more hyperpolarized potentials. Immunofluorescence labeling and western immunoblotting with an anti-NaV1.1 antibody, revealed that BIL elevates the expression of VGSCs by promoting their recruitment to the membrane. Furthermore, we found that this VGSC-trafficking process is Ca2+ dependent because preloading MVN neurons with the Ca2+ buffer BAPTA-AM, or exocytosis inhibitor TAT-NSF700, prevents the effects of BIL, indicating the upregulated activity and density of functional VGSCs as the core mechanism accountable for the BIL-induced overexcitation of neonatal neurons. Most importantly, rectification of such overexcitation with a low dose of VGSC blocker lidocaine significantly attenuates BIL-induced cell death. We suggest that this enhancement of VGSC currents directly contributes to the vulnerability of neonatal brain to hyperbilirubinemia, implicating the activity and trafficking of NaV1.1 channels as a potential target for neuroprotection in cases of severe jaundice.

The General Anesthetic Isoflurane Bilaterally Modulates Neuronal Excitability

Volatile anesthetics induce hyperactivity during induction while produce anesthesia at higher concentrations. They also bidirectionally modulate many neuronal functions. However, the neuronal mechanism is quite unclear. The effects of isoflurane on sodium channel currents were analyzed in acute mouse brain slices, including sodium leak (NALCN) currents, and voltage-gated sodium channels (Nav) currents. Isoflurane at sub-anesthetic concentrations increased the spontaneous firing rate of CA3 pyramidal neurons, while anesthetic concentrations of isoflurane decreased the firing rate. Isoflurane at sub-anesthetic concentrations enhanced NALCN conductance, but minimally inhibited Nav currents. Isoflurane at anesthetic concentrations depressed Nav currents and action potential amplitudes. Isoflurane at sub-MAC concentrations depolarized resting membrane potential (RMP) of neurons while hyperpolarized the RMP at anesthetic concentrations. Isoflurane at low concentrations induced hyperactivity in vivo, which was diminished in NALCN knockdown mice. In conclusion, enhancement of NALCN by isoflurane contributes to its bidirectional modulation of neuronal excitability and the hyperactivity during induction.

Tetrodotoxin-Sensitive Sodium Channels Mediate Action Potential Firing and Excitability in Menthol-Sensitive Vglut3-Lineage Sensory Neurons.

Small-diameter vesicular glutamate transporter 3-lineage (Vglut3lineage) dorsal root ganglion (DRG) neurons play an important role in mechanosensation and thermal hypersensitivity; however, little is known about their intrinsic electrical properties. We therefore set out to investigate mechanisms of excitability within this population. Calcium microfluorimetry analysis of male and female mouse DRG neurons demonstrated that the cooling compound menthol selectively activates a subset of Vglut3lineage neurons. Whole-cell recordings showed that small-diameter Vglut3lineage DRG neurons fire menthol-evoked action potentials and exhibited robust, transient receptor potential melastatin 8 (TRPM8)-dependent discharges at room temperature. This heightened excitability was confirmed by current-clamp and action potential phase-plot analyses, which showed menthol-sensitive Vglut3lineage neurons to have more depolarized membrane potentials, lower firing thresholds, and higher evoked firing frequencies compared with menthol-insensitive Vglut3lineage neurons. A biophysical analysis revealed voltage-gated sodium channel (NaV) currents in menthol-sensitive Vglut3lineage neurons were resistant to entry into slow inactivation compared with menthol-insensitive neurons. Multiplex in situ hybridization showed similar distributions of tetrodotoxin (TTX)-sensitive NaV transcripts between TRPM8-positive and -negative Vglut3lineage neurons; however, NaV1.8 transcripts, which encode TTX-resistant channels, were more prevalent in TRPM8-negative neurons. Conversely, pharmacological analyses identified distinct functional contributions of NaV subunits, with NaV1.1 driving firing in menthol-sensitive neurons, whereas other small-diameter Vglut3lineage neurons rely primarily on TTX-resistant NaV channels. Additionally, when NaV1.1 channels were blocked, the remaining NaV current readily entered into slow inactivation in menthol-sensitive Vglut3lineage neurons. Thus, these data demonstrate that TTX-sensitive NaVs drive action potential firing in menthol-sensitive sensory neurons and contribute to their heightened excitability.SIGNIFICANCE STATEMENT Somatosensory neurons encode various sensory modalities including thermoreception, mechanoreception, nociception, and itch. This report identifies a previously unknown requirement for tetrodotoxin-sensitive sodium channels in action potential firing in a discrete subpopulation of small-diameter sensory neurons that are activated by the cooling agent menthol. Together, our results provide a mechanistic understanding of factors that control intrinsic excitability in functionally distinct subsets of peripheral neurons. Furthermore, as menthol has been used for centuries as an analgesic and anti-pruritic, these findings support the viability of NaV1.1 as a therapeutic target for sensory disorders.

NMDA Receptors Regulate Neuregulin 2 Binding to ER-PM Junctions and Ectodomain Release by ADAM10 [corrected

Unprocessed pro-neuregulin 2 (pro-NRG2) accumulates on neuronal cell bodies at junctions between the endoplasmic reticulum and plasma membrane (ER-PM junctions). NMDA receptors (NMDARs) trigger NRG2 ectodomain shedding from these sites followed by activation of ErbB4 receptor tyrosine kinases, and ErbB4 signaling cell-autonomously downregulates intrinsic excitability of GABAergic interneurons by reducing voltage-gated sodium channel currents. NMDARs also promote dispersal of Kv2.1 clusters from ER-PM junctions and cause a hyperpolarizing shift in its voltage-dependent channel activation, suggesting that NRG2/ErbB4 and Kv2.1 work together to regulate intrinsic interneuron excitability in an activity-dependent manner. Here we explored the cellular processes underlying NMDAR-dependent NRG2 shedding in cultured rat hippocampal neurons. We report that NMDARs control shedding by two separate but converging mechanisms. First, NMDA treatment disrupts binding of pro-NRG2 to ER-PM junctions by post-translationally modifying conserved Ser/Thr residues in its intracellular domain. Second, using a mutant NRG2 protein that cannot be modified at these residues and that fails to accumulate at ER-PM junctions, we demonstrate that NMDARs also directly promote NRG2 shedding by ADAM-type metalloproteinases. Using pharmacological and shRNA-mediated knockdown, and metalloproteinase overexpression, we unexpectedly find that ADAM10, but not ADAM17/TACE, is the major NRG2 sheddase acting downstream of NMDAR activation. Together, these findings reveal how NMDARs exert tight control over the NRG2/ErbB4 signaling pathway, and suggest that NRG2 and Kv2.1 are co-regulated components of a shared pathway that responds to elevated extracellular glutamate levels.

Capsazepine prolongation of the duration of lidocaine block of sensory transmission in mice may be mediated by modulation of HCN channel currents.

Background and objectivesHyperpolarization-activation cyclic nucleotide-gated (HCN) channels contribute to the effects of lidocaine. Capsazepine (CPZ), a competitive inhibitor of capsaicin of transient receptor potential vanilloid-1 channel, has also been found to inhibit HCN channel currents (Ih). This study was designed to investigate whether CPZ could prolong durations of lidocaine in regional anesthesia.MethodsMouse HCN1 and HCN2 channels were expressed in human embryonic kidney 293 (HEK 293) cells. The effect of CPZ on Ih was measured by whole-cell patch-clamping recording. Sciatic nerve block model in mice was used for the study in vivo. The mice were randomly divided into seven groups, respectively, receiving lidocaine, CPZ, ZD7288 (HCN channel blocker), CPZ + lidocaine, ZD7288 + lidocaine, ZD7288 + CPZ + lidocaine, forskolin (an activator of adenylyl cyclase) + CPZ + lidocaine. Regional anesthetic durations of lidocaine were determined. Voltage-gated sodium channel currents (INa) and Ih were recorded in dorsal root ganglion neurons of mice. The effects of CPZ on INa and Ih with or without Cyclic adenosine monophosphate (cAMP) were assessed. Isolated mice sciatic nerve was prepared to evaluate the effect of CPZ on the compound action potentials (CAP).ResultsCapsazepine non-selectively inhibited transfected mHCN1 and mHCN2 channel currents in HEK 293 cells. In sciatic nerve block in vivo, compared to lidocaine alone, adding CPZ extended the durations of lidocaine for noxious sensory block (35.1 ± 3.3 vs. 20.3 ± 1.7 min), tactile sensory block (25.5 ± 4.4 vs. 20.0 ± 3.7 min), thermal sensory block (39.6 ± 6.6 vs. 26.8 ± 5.5 min), and motor function block (28.6 ± 4.1 vs. 20.9 ± 4.2 min). Duration of thermal sensory block was longer in CPZ + lidocaine group than that of ZD7288 + lidocaine group (39.6 ± 6.6 vs. 33.4 ± 4.5 min). Forskolin reversed the prolongation by CPZ on lidocaine durations. CPZ or ZD7288 alone did not produce typical regional anesthetic effects. Increased intracellular concentration of cAMP reversed the inhibition of CPZ on Ih. Although CPZ alone inhibited INa at the concentration more than 30 μM, it did not inhibit the CAP amplitudes in isolated sciatic nerves. CPZ dose-dependently enhanced the inhibitory effect of 1% lidocaine on the CAP amplitudes.ConclusionsCapsazepine may prolong durations of lidocaine in peripheral nerve block by modulation of HCN channel currents.

Dehydrocrenatidine Inhibits Voltage-Gated Sodium Channels and Ameliorates Mechanic Allodia in a Rat Model of Neuropathic Pain.

Picrasma quassioides (D. Don) Benn, a medical plant, is used in clinic to treat inflammation, pain, sore throat, and eczema. The alkaloids are the main active components in P. quassioides. In this study, we examined the analgesic effect of dehydrocrenatidine (DHCT), a β-carboline alkaloid abundantly found in P. quassioides in a neuropathic pain rat model of a sciatic nerve chronic constriction injury. DHCT dose-dependently attenuated the mechanic allodynia. In acutely isolated dorsal root ganglion, DHCT completely suppressed the action potential firing. Further electrophysiological characterization demonstrated that DHCT suppressed both tetrodotoxin-resistant (TTX-R) and sensitive (TTX-S) voltage-gated sodium channel (VGSC) currents with IC50 values of 12.36 μM and 4.87 µM, respectively. DHCT shifted half-maximal voltage (V1/2) of inactivation to hyperpolarizing direction by ~16.7 mV in TTX-S VGSCs. In TTX-R VGSCs, DHCT shifted V1/2 of inactivation voltage to hyperpolarizing direction and V1/2 of activation voltage to more depolarizing potential by ~23.9 mV and ~12.2 mV, respectively. DHCT preferred to interact with an inactivated state of VGSCs and prolonged the repriming time in both TTX-S and TTX-R VGSCs, transiting the channels into a slow inactivated state from a fast inactivated state. Considered together, these data demonstrated that the analgesic effect of DHCT was likely though the inhibition of neuronal excitability.

Dexmedetomidine Inhibits Voltage-Gated Sodium Channels via α2-Adrenoceptors in Trigeminal Ganglion Neurons.

Dexmedetomidine, an α2-adrenoceptor agonist, is widely used as a sedative and analgesic agent in a number of clinical applications. However, little is known about the mechanism by which it exerts its analgesic effects on the trigeminal system. Two types of voltage-gated sodium channels, Nav1.7 and Nav1.8, as well as α2-adrenoceptors are expressed in primary sensory neurons of the trigeminal ganglion (TG). Using whole-cell patch-clamp recordings, we investigated the effects of dexmedetomidine on voltage-gated sodium channel currents (INa) via α2-adrenoceptors in dissociated, small-sized TG neurons. Dexmedetomidine caused a concentration-dependent inhibition of INa in small-sized TG neurons. INa inhibition by dexmedetomidine was blocked by yohimbine, a competitive α2-adrenoceptor antagonist. Dexmedetomidine-induced inhibition of INa was mediated by G protein-coupled receptors (GPCRs) as this effect was blocked by intracellular perfusion with the G protein inhibitor GDPβ-S. Our results suggest that the INa inhibition in small-sized TG neurons, mediated by the activation of Gi/o protein-coupled α2-adrenoceptors, might contribute to the analgesic effects of dexmedetomidine in the trigeminal system. Therefore, these new findings highlight a potential novel target for analgesic drugs in the orofacial region.

Protein Kinase C Mediates the Corticosterone-induced Sensitization of Dorsal Root Ganglion Neurons Innervating the Rat Stomach.

Background/AimsGastric hypersensitivity contributes to abdominal pain in patients with functional dyspepsia. Recent studies showed that hormones induced by stress are correlated with visceral hypersensitivity. However, the precise mechanisms underlying gastric hypersensitivity remain largely unknown. The aim of the present study was designed to investigate the roles of corticosterone (CORT) on excitability of dorsal root ganglion (DRG) neurons innervating the stomach.MethodsDRG neurons innervating the stomach were labeled by DiI injection into the stomach wall. Patch clamp recordings were employed to examine neural excitability and voltage-gated sodium channel currents. Electromyograph technique was used to determine the responses of neck muscles to gastric distension.ResultsIncubation of acutely isolated DRG neurons with CORT significantly depolarized action potential threshold and enhanced the number of action potentials induced by current stimulation of the neuron. Under voltage-clamp mode, incubation of CORT enhanced voltage-gated sodium current density of the recorded neurons. Pre-incubation of GF109203X, an inhibitor of protein kinase C, blocked the CORT-induced hyperexcitability and potentiation of sodium currents. However, pre-incubation of H-89, an inhibitor of protein kinase A, did not alter the sodium current density. More importantly, intraperitoneal injection of CORT produced gastric hypersensitivity of healthy rats, which was blocked by pre-administration of GF109203X but not H-89.ConclusionsOur data strongly suggest that CORT rapidly enhanced neuronal excitability and sodium channel functions, which is most likely mediated by protein kinase C but not protein kinase A signaling pathway in DRG neurons innervating the stomach, thus underlying the gastric hypersensitivity induced by CORT injection.