Action Potential Firing Research Articles

Camphor Attenuates Hyperalgesia in Neuropathic Pain Models in Mice.

The treatment of neuropathic pain is still a majortroublesomeclinicalproblem. The existing therapeutic drugs have limited analgesic effect and obvious adverse reactions, which presents opportunities and challenges for the development of new analgesic drugs. Camphor, a kind of monoterpene, has been shown anti-inflammatory and analgesic effects in traditional Chinese medicine. But we know little about its effect in neuropathic pain. In this article, We have verified the reliable analgesic effect of camphor in the neuropathic pain model caused by different predispositions. The nociceptive response of mice was induced by transient receptor potential A1 (TRPA1) agonist to verify the effect of camphor on the nociceptive response. Multiple paclitaxel (PTX) injection models, Single oxaliplatin (OXA) injection models, Chronic constriction injury (CCI) models and Streptozotocin-induced (STZ) diabetic neuropathic pain models were used in this study. We verified the analgesic effect of camphor in mice by acetone test and conditioned place aversion test. At the same time, comparing the adverse reaction of nervous system between camphor and pregabalin at equivalent dose in locomotor activity test and rotarod test. Using patch clamp to verify the effect of camphor on dorsal root ganglion (DRG) excitability. In behavioral test, compared with vehicle group, camphor significantly reduced the spontaneous nociception caused by TRPA1 agonist-formalina and allyl isothiocyanate (AITC). Compared with vehicle group, camphor significantly reduced the flinching and licking time in neuropathic pain model mice, including PTX, OXA, STZ and CCI induced peripheral neuralgia models. Compared with vehicle group, pregabalin significantly increased the resting time and reduced the average speed without resting and distance in locomotor activity test, reduced the time stayed on rotarod in rotarod test. In patch clamp test, compared with vehicle group, camphor significantly reduced the action potential (AP) firing frequency of DRG. Camphor can alleviate the symptoms of hyperalgesia in various neuropathic pain models, and has no obvious adverse reactions compared with pregabalin. This effect is related to the down-regulation of DRG neuron excitability.

Plateau potentials contribute to myotonia in mouse models of myotonia congenita.

It has long been accepted that myotonia (muscle stiffness) in patients with muscle channelopathies is due to myotonic discharges (involuntary firing of action potentials). In a previous study, we identified a novel phenomenon in myotonic muscle: development of plateau potentials, transient depolarizations to near -35mV lasting for seconds to minutes. In the current study we examined whether plateau potentials contribute to myotonia. A recessive genetic model (ClCadr mice) with complete loss of muscle chloride channel (ClC-1) function was used to model severe myotonia congenita with complete loss of ClC-1 function and a pharmacologic model using anthracene-9-carboxylic acid (9AC) was used to model milder myotonia congenita with incomplete loss of ClC-1 function. Simultaneous measurements of action potentials and myoplasmic Ca2+ from individual muscle fibers were compared to recordings of whole muscle force generation. In ClCadr muscle both myotonia and plateau potentials lasted 10s of seconds to minutes. During plateau potentials lasting 1-2min, there was a gradual transition from high to low intracellular Ca2+, suggesting a transition in individual fibers from myotonia to flaccid paralysis in severe myotonia congenita. In 9AC-treated muscles, both myotonia and plateau potentials lasted only a few seconds and Ca2+ remained elevated during the plateau potentials, suggesting plateau potentials contribute to myotonia without causing weakness. We propose, that in myotonic muscle, there is a novel state in which there is contraction in the absence of action potentials. This discovery provides a mechanism to explain reports of patients with myotonia who suffer from electrically silent muscle contraction lasting minutes.

Aluminum alters excitability by inhibiting calcium, sodium, and potassium currents in bovine chromaffin cells.

Aluminum (Al3+ ) has long been related to neurotoxicity and neurological diseases. This study aims to describe the specific actions of this metal on cellular excitability and neurotransmitter release in primary culture of bovine chromaffin cells. Using voltage-clamp and current-clamp recordings with the whole-cell configuration of the patch clamp technique, online measurement of catecholamine release, and measurements of [Ca2+ ]c with Fluo-4-AM, we have observed that Al3+ reduced intracellular calcium concentrations around 25% and decreased catecholamine secretion in a dose-dependent manner, with an IC50 of 89.1μM. Al3+ blocked calcium currents in a time- and concentration-dependent manner with an IC50 of 560 μM. This blockade was irreversible since it did not recover after washout. Moreover, Al3+ produced a bigger blockade on N-, P-, and Q-type calcium channels subtypes (69.5%) than on L-type channels subtypes (50.5%). Sodium currents were also inhibited by Al3+ in a time- and concentration-dependent manner, 24.3% blockade at the closest concentration to the IC50 (399 μM). This inhibition was reversible. Voltage-dependent potassium currents were low affected by Al3+ . Nonetheless, calcium/voltage-dependent potassium currents were inhibited in a concentration-dependent manner, with an IC50 of 447 μM. This inhibition was related to the depression of calcium influx through voltage-dependent calcium channels subtypes coupled to BK channels. In summary, the blockade of these ionic conductance altered cellular excitability that reduced the action potentials firing and so, the neurotransmitter release and the synaptic transmission. These findings prove that aluminum has neurotoxic properties because it alters neuronal excitability by inhibiting the sodium currents responsible for the generation and propagation of impulse nerve, the potassium current responsible for the termination of action potentials, and the calcium current responsible for the neurotransmitters release.

Oxytocin mediated excitation of hypoglossal motoneurons: implications for treating obstructive sleep apnea.

Clinical studies have shown that oxytocin administered intranasally (IN) decreased the incidence and duration of obstructive events in patients with obstructive sleep apnea (OSA). Although the mechanisms by which oxytocin promotes these beneficial effects are unknown, one possible target of oxytocin could be the excitation of tongue-projecting hypoglossal motoneurons in the medulla, that exert central control of upper airway patency. This study tested the hypothesis that IN oxytocin enhances tongue muscle activity via the excitation of hypoglossal motoneurons projecting to tongue protrudor muscles (PMNs). To test this hypothesis we performed in vivo and in vitro electrophysiological studies in C57BL6/J mice as well as fluorescent imaging studies in transgenic mice in which neurons that express oxytocin receptors co-express fluorescent protein. IN oxytocin significantly increased the amplitude of inspiratory-related tongue muscle activity. This effect was abolished by severing the medial branch of hypoglossal nerve that innervates PMNs of the tongue. Oxytocin receptor-positive neurons were more prevalent in the population of PMNs than in retractor-projecting hypoglossal motoneurons (RMNs). Oxytocin administration increased action potential firing in PMNs, but had no significant effect on firing activity in RMNs. In conclusion, IN oxytocin stimulates respiratory-relating tongue muscle activity likely acting on central hypoglossal motoneurons that provide tongue protrusion and upper airway opening. This mechanism may play a role in oxytocin-induced reductions in upper airway obstructions in patients with OSA.

LGI3/2-ADAM23 interactions cluster Kv1 channels in myelinated axons to regulate refractory period.

Along myelinated axons, Shaker-type potassium channels (Kv1) accumulate at high density in the juxtaparanodal region, directly adjacent to the paranodal axon-glia junctions that flank the nodes of Ranvier. However, the mechanisms that control the clustering of Kv1 channels, as well as their function at this site, are still poorly understood. Here we demonstrate that axonal ADAM23 is essential for both the accumulation and stability of juxtaparanodal Kv1 complexes. The function of ADAM23 is critically dependent on its interaction with its extracellular ligands LGI2 and LGI3. Furthermore, we demonstrate that juxtaparanodal Kv1 complexes affect the refractory period, thus enabling high-frequency burst firing of action potentials. Our findings not only reveal a previously unknown molecular pathway that regulates Kv1 channel clustering, but they also demonstrate that the juxtaparanodal Kv1 channels that are concealed below the myelin sheath, play a significant role in modifying axonal physiology.

Small molecules fail to induce direct reprogramming of adult rat olfactory ensheathing glia to mature neurons.

An approach to generate new neurons after central nervous system injury or disease is direct reprogramming of the individual's own somatic cells into differentiated neurons. This can be achieved either by transduction of viral vectors that express neurogenic transcription factors and/or through induction with small molecules, avoiding introducing foreign genetic material in target cells. In this work, we propose olfactory ensheathing glia (OEG) as a candidate for direct reprogramming to neurons with small molecules due to its well-characterized neuro-regenerative capacity. After screening different combinations of small molecules in different culture conditions, only partial reprogramming was achieved: induced cells expressed neuronal markers but lacked the ability of firing action potentials. Our work demonstrates that direct conversion of adult olfactory ensheathing glia to mature, functional neurons cannot be induced only with pharmacological tools.

Osmotically evoked PLCδ1-dependent translocation of ΔN-TRPV1 channels in rat supraoptic neurons

Osmoregulation is an essential homeostatic process that requires constant release of vasopressin during sustained increases in plasma osmolality. The magnocellular neurosecretory cells (MNCs) respond to increases in external osmolality through increases in the activity of ΔN-TRPV1 channels, which leads to increased action potential firing and vasopressin release. We show that sustained exposure of acutely isolated rat and mouse MNCs to hypertonic solutions (90min) causes a reversible translocation of ΔN-TRPV1 channels from internal stores to the plasma membrane that depends on the activation of phospholipase C and on SNARE-dependent exocytosis. ΔN-TRPV1 channel translocation is absent in MNCs isolated from transgenic mice lacking the PLCδ1 isoform, suggesting that PLCδ1 is essential for triggering this process. The translocation of ΔN-TRPV1 channels to the cell surface could contribute to the maintenance of MNC excitability during sustained increases in osmolality. Our data therefore have important implications for the mechanisms underlying mammalian osmoregulation.

Lobule-Related Action Potential Shape- and History-Dependent Current Integration in Purkinje Cells of Adult and Developing Mice.

Purkinje cells (PCs) are the principal cells of the cerebellar cortex and form a central element in the modular organization of the cerebellum. Differentiation of PCs based on gene expression profiles revealed two subpopulations with distinct connectivity, action potential firing and learning-induced activity changes. However, which basal cell physiological features underlie the differences between these subpopulations and to what extent they integrate input differentially remains largely unclear. Here, we investigate the cellular electrophysiological properties of PC subpopulation in adult and juvenile mice. We found that multiple fundamental cell physiological properties, including membrane resistance and various aspects of the action potential shape, differ between PCs from anterior and nodular lobules. Moreover, the two PC subpopulations also differed in the integration of negative and positive current steps as well as in size of the hyperpolarization-activated current. A comparative analysis in juvenile mice confirmed that most of these lobule-specific differences are already present at pre-weaning ages. Finally, we found that current integration in PCs is input history-dependent for both positive and negative currents, but this is not a distinctive feature between anterior and nodular PCs. Our results support the concept of a fundamental differentiation of PCs subpopulations in terms of cell physiological properties and current integration, yet reveals that history-dependent input processing is consistent across PC subtypes.

T-type calcium channels as therapeutic targets in essential tremor and Parkinson's disease.

Neuronal action potential firing patterns are key components of healthy brain function. Importantly, restoring dysregulated neuronal firing patterns has the potential to be a promising strategy in the development of novel therapeutics for disorders of the central nervous system. Here, we review the pathophysiology of essential tremor and Parkinson's disease, the two most common movement disorders, with a focus on mechanisms underlying the genesis of abnormal firing patterns in the implicated neural circuits. Aberrant burst firing of neurons in the cerebello-thalamo-cortical and basal ganglia-thalamo-cortical circuits contribute to the clinical symptoms of essential tremor and Parkinson's disease, respectively, and T-type calcium channels play a key role in regulating this activity in both the disorders. Accordingly, modulating T-type calcium channel activity has received attention as a potentially promising therapeutic approach to normalize abnormal burst firing in these diseases. In this review, we explore the evidence supporting the theory that T-type calcium channel blockers can ameliorate the pathophysiologic mechanisms underlying essential tremor and Parkinson's disease, furthering the case for clinical investigation of these compounds. We conclude with key considerations for future investigational efforts, providing a critical framework for the development of much needed agents capable of targeting the dysfunctional circuitry underlying movement disorders such as essential tremor, Parkinson's disease, and beyond.

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.

Optical modulation of excitation-contraction coupling in human-induced pluripotent stem cell-derived cardiomyocytes

Non-genetic photostimulation is a novel and rapidly growing multidisciplinary field that aims to induce light-sensitivity in living systems by exploiting exogeneous phototransducers. Here, we propose an intramembrane photoswitch, based on an azobenzene derivative (Ziapin2), for optical pacing of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The light-mediated stimulation process has been studied by applying several techniques to detect the effect on the cell properties. In particular, we recorded changes in membrane capacitance, in membrane potential (Vm), and modulation of intracellular Ca2+ dynamics. Finally, cell contractility was analyzed using a custom MATLAB algorithm. Photostimulation of intramembrane Ziapin2 causes a transient Vm hyperpolarization followed by a delayed depolarization and action potential firing. The observed initial electrical modulation nicely correlates with changes in Ca2+ dynamics and contraction rate. This work represents the proof of principle that Ziapin2 can modulate electrical activity and contractility in hiPSC-CMs, opening up a future development in cardiac physiology.

Action potential firing and sensory transduction are sustained by membrane potential instabilities in peripheral sensory neurons.

Peripheral sensory neurons are fundamental for stimulus detection, but many of their properties remain poorly understood (Velasco et al., 2022). One of these is the marked instability of the resting membrane potential recorded in such neurons, known as Membrane Potential Instabilities (MPIs). MPIs are enhanced in pain states, but their functional significance and molecular bases remain largely unknown. Here, we addressed this using whole-cell current-clamp recordings in primary cultured mouse trigeminal (TG) neurons. MPI detection and morphological analyses were performed semi-automatically, using custom-made scripts. We found that 82.2% of small diameter neurons recorded displayed MPI activity in the shape of Membrane Potential Transients (MPTs), spontaneously or upon depolarizing current injection. The frequency, amplitude and rates of upstroke and repolarization of MPTs were increased by membrane depolarization. Morphological analyses of the MPTs and of the initial depolarizing phase of action potentials (Aps) revealed that larger and faster MPTs precede AP firing. This was confirmed by a regression analysis showing that the maximal rate of depolarization and amplitude of the MPTs strongly predict subsequent AP firing, following a Boltzmann dependency. MPT activity was enhanced by the application of heat, cold, capsaicin and menthol, via their depolarizing effect on the resting membrane potential. MPT activity was reduced by blocking TTX-resistant, but not TTX-sensitive voltage-gated Na+ channels. Accordingly, MPTs were also reduced in neurons isolated from NaV1.8 and NaV1.9 knockout mice. We hereby identify, for the first time, the molecular determinants of the trigger of MPTs and demonstrate a causal relationship between MPTs and AP firing upon exogenous stimulation of sensory neurons. These findings unveil MPTs as crucial regulators of peripheral sensory processing, with possible implications in pathological conditions featuring altered activities of TTX-resistant NaV channels.

Genetic deletion of STAC2 adaptor protein alters electrical activity of mouse chromaffin cells.

Voltage-gated Cav1.2 and Cav1.3 L-type Ca2+ channels (LTCCs) regulate action potential (AP) firing and catecholamine release in mouse chromaffin cells (MCCs). Src homology 3 and cysteine rich domain adaptor proteins (Stac1-Stac3) have recently been identified as novel regulators of neuronal LTCCs expression and biophysical properties. Upon overexpression in cultured hippocampal neurons Stac2 abolished LTCC Ca2+ dependent inactivation via an allosteric inhibition of calmodulin binding. Additionally, in Drosophila, deletion of the homologous DStac gene resulted in deficient LTCC Ca2+ transients and reduced neuropeptide release. Here we investigated the effect of Stac2 genetic ablation on neuronal LTCC function, electrical activity, and vesicle exocytosis in MCCs. Constitutive deletion of Stac2 does not affect the MCCs resting membrane potential or spontaneous firing frequency. However, the AP depolarization threshold was significantly reduced in Stac2-/- MCCs compared to WT (WT = −35.6 ± 1.2 mV, Stac2-/- = −40.6 ± 0.6 mV; ∗∗ P < 0.01). Additionally, step current injection elicited an electrical activity with higher initial AP firing frequency in Stac2-/- compared to WT that led to earlier depolarization block (F0 ≅2 pA current injection: WT = 0.28 ± 0.28 Hz, Stac2-/- = 0.89 ± 0.40 Hz; ∗ P < 0.05). Surprisingly, initial characterization shows that Stac2 deletion does not alter whole-cell Ca2+ current amplitude or inactivation kinetics but significantly shifts the voltage-dependence of activation to more hyperpolarized potential (V0.5: WT = −5.95 ± 1.65 mV, Stac2-/- = −15.25 ± 2.05 mV; ∗ P < 0.05). We are currently investigating possible effects of Stac2 deletion on other ionic conductances governing MCC excitability and the role on catecholamine vesicle exocytosis. Support: TWF F.18863, LFU 2021-CHEM-8 to SMG; FWF P31434, P36053, DOC30-B30 to PT; P33776 to MC.

Defining the interactions between HCN4 and the regulatory proteins IRAG and LRMP.

Hyperpolarization-activated, cyclic nucleotide-sensitive HCN4 channels are the molecular determinants of the funny (If) current, a mixed Na+/K+ current that controls pacemaking in cardiac cells. In the sinoatrial node (SAN), an increase in If in response to cAMP contributes to an increase in action potential (AP) firing rate and consequently heart rate. We recently discovered two related modulators of HCN4 function: lymphoid-restricted membrane protein (LRMP) and inositol trisphosphate receptor-associated guanylate kinase substrate (IRAG). Despite homology between these two proteins, LRMP inhibits the depolarizing shift in the voltage dependence of activation of HCN4 in response to cAMP while IRAG shifts the basal voltage dependence in the depolarizing direction. Even with a clear picture of the functional effects that these two proteins exert on HCN4, the domains responsible for the interactions are completely unknown. Here, we pair a FRET two-hybrid approach with patch clamp electrophysiology to determine which domains of LRMP, IRAG, and HCN4 are involved in complex formation. Both LRMP and IRAG have small ER luminal domains, ER transmembrane segments, and a large cytoplasmic domain that likely interacts with an intracellular portion of HCN4 channels. By co-expressing cerulean-tagged cytosolic fragments of HCN4 with citrine-tagged fragments of LRMP and IRAG, we determine which regions on each protein are required for complex formation using FRET. We then test the functional effect of removing these critical domains on the regulation of the channel by each protein using patch clamp electrophysiology. Our preliminary results suggest that the N-terminus of HCN4 is critical for both LRMP and IRAG regulation of the channel. Moreover, the first 230 amino acids of LRMP is sufficient to bind HCN4. These results shed light on a novel amino terminus-dependent mechanism for regulating the gating of HCN4 channels.

Kv1 activator mechanism of action in action potential inhibition.

Delayed-rectifier, voltage-gated potassium channels (Kv), such as the Kv1 family, are found in nociceptive dorsal root ganglion (DRG) neurons and represent a potential target for novel analgesics. Activating Kv1 channels in nociceptive neurons may work to decrease neuronal excitability and diminish the sensation of pain. Here, we explored the mechanism by which a Kv1 activator decreases action potential firing in DRG neurons. The small molecules compound A (cmpd-A) and B (cmpd-B) induced a hyperpolarizing shift in the conductance-voltage relationship of a heterologously-expressed Kv1 channel while cmpd-A additionally slowed the deactivation rate in a voltage-independent manner. Cmpd-A, but not cmpd-B, caused a frequency-dependent inhibition of AP firing in rat DRG neurons. We examined the effects of both compounds on Kv1 biophysics and neuronal membrane properties to explain the mechanism of action underlying cmpd-A AP inhibition.

Patch clamp study of benzydamine effect over neuronal excitability on inflammation sensitized rat nociceptors.

Benzydamine (BZ) is an active principle with local anti-inflammatory effect and analgesic properties widely used in oral affections. BZ mechanism of action has been deeply characterized on inflammatory cell types and mediators, highlighting its capacity to inhibit pro-inflammatory mediator's synthesis and release. Nevertheless, its pharmacological action as analgesic has been slightly explored, even more in inflammatory condition, and a more precise understanding of its action at single cell level is needed. To better understand BZ effect on isolated nociceptors, single cell neuronal activity was studied by patch clamp after acute or chronic inflammatory sensitization. Consequently, DRG nociceptors were challenged either by 5-minutes or 24-hours incubation with an inflammatory soup (IS - containing histamine, serotonin, ATP and PGE2), or BZ, or simultaneously with IS and BZ. BZ acute application demonstrated a decrease in action potential firing frequency supported also by rheobase increase and afterhyperpolarization (AHP) amplitude increase. Acute BZ application simultaneously with IS, limited neuronal sensitization contrary to the effect observed after IS application alone, which increased twice the action potential firing frequency. Moreover, chronic incubation of BZ tended to protect neurons against inflammatory sensitization. These results reinforce a previous MEA study, demonstrating a reduction of neuronal excitability elicited by inflammatory mediators after BZ use.As a conclusion we hypothesize that the observed decrease in firing frequency and rheobase increase could be related to the direct inhibition of Nav 1.8 and 1.9 channels, while the changes in AHP could relate with HCN or Kv7 channels modulation. An action via proalgesic channels like ASIC or TRP, sensitized by the inflammatory environment, cannot be excluded. Overall results reinforce the analgesic and anti-inflammatory effect of benzydamine, supporting the complementary action of the compound on local pain signalling and inflammatory conditions.

SCN1A gain of function causes interneuron hyperexcitability and network instability through inhibitory synaptic plasticity in early infantile encephalopathy.

SCN1A gain-of-function variants are associated with early infantile and neonatal-onset developmental and epileptic encephalopathies (DEE). It is unclear how SCN1A gain-of-function may predispose to cortical hyper-excitability and seizures. We studied the biophysical properties of four pathogenic Nav1.1 variants associated with neonatal-onset (T162I, I236V) or early infantile (P1345S, R1636Q) DEE. In voltage clamp experiments, the activation curves of the variants were either unchanged (T236V), or exhibited hyperpolarized shifts (T162I, P1345S, R1636Q), corresponding to gain-of-function through enhanced window currents. Inactivation curves were unchanged (I236V, P1345S), depolarized, suggesting gain-of-function (R1636Q), or hyperpolarized, suggesting loss-of-function (T162I). Dynamic action potential clamp experiments supported a gain of function mechanism for all variants. Model neurons incorporating Nav1.1. channels with properties of I236V, P1345S, or R1636Q variants exhibited higher and sustained firing relative to wild type, whereas T162I exhibited enhanced early action potential firing and depolarization block. T162I and R1636Q produced a hyperpolarized threshold and reduced neuronal rheobase. To explore the impact of SCN1A variants upon cortical excitability, we used a cortical network model containing excitatory pyramidal cells, PV interneurons and realistic synaptic connectivity. SCN1A gain-of-function was modelled by enhancing PV excitability and incorporating a simple form of homeostatic adaptation: inhibitory synaptic plasticity. Although both wild-type and gain-of-function networks possessed similar basal pyramidal cell firing rates, the gain-of-function network was more sensitive to external perturbation and exhibited synchronous epileptiform-like activity. Further analysis revealed that reduced synaptic strength results in a deficit of feedback inhibition and an increased excitatory-inhibitory ratio in the gain-of-function network. Overall, our findings support the role of SCN1A gain-of-function in early onset DEE. We propose a mechanism through which homeostatic adaptations in neuronal networks can predispose to enhanced excitatory activity and seizures.

Calcium inhibits the funny current (If)in sinoatrial myocytes.

Hyperpolarization-activated cyclic nucleotide sensitive (HCN4) channels are the molecular correlate of the funny current (If), which is required for spontaneous action potentials in cardiac pacemaker cells. Activation of β-adrenergic receptors on pacemaker cells increases cAMP, which shifts HCN4 activation to more depolarized potentials. The resulting increase in current, along with activation of Ca2+ cycling proteins, increases action potential firing rate and, thus, heart rate. Here we show that Mg2+ and Ca2+ regulate the effects of cAMP on If and HCN4. We found that physiological concentrations of intracellular Ca2+ inhibited cAMP-dependent potentiation of native If channels in excised membrane patches from sinoatrial node myocytes (IC50 ≈ 9 μM). This inhibition was recapitulated in heterologously expressed HCN4 channels, where intracellular Ca2+ inhibited the channels with a similar IC50 (≈ 5 μM) and limited the cAMP-dependent shift in channel activation (4 mV in 100 µM Ca2+ vs. 12 mV in zero Ca2+). Interestingly, Mg2+ had opposing effects to Ca2+. Mg2+ has been shown to interact with cAMP-bound HCN4 channels at a metal coordination site composed of two residues in the C-linker and two residues in the S4-S5 linker. We found that Mg2+ is also required to functionally couple the inhibitory, unbound CNBD to the pore in the absence of cAMP — removal of Mg2+ mimicked the effects of cAMP-dependent disinhibition. Although the metal coordination site is conserved among mammalian HCN channel isoforms, Mg2+ and Ca2+ regulation appear to be specific for HCN4 — cAMP regulation of HCN2 was insensitive to divalents. These results suggest that divalent cations directly regulate HCN4, raising the possibility that If in pacemaker cells is dynamically regulated in response to beat-to-beat changes in intracellular Ca2+.

Increased pancreatic β-cell electrical activity reduces diabetes susceptibility in female mice.

In humans, the incidence of Type 2 Diabetes Mellitus (T2DM) in premenopausal women is higher compared to men, phenotype also recapitulated by many rodent models. The T2DM etiology involves impaired or total lack of insulin release from pancreatic β-cells. High voltage-gated Ca2+ channels (HVCCs) are multi-subunit protein complexes responsible for β-cell electrical activity and insulin vesicle exocytosis. Previously, we showed that genetic deletion of α2δ-1 HVCC auxiliary subunit equally reduced Ca2+ influx by 60% in β-cells of both males and females but caused diabetes only in males because female pancreatic islets released significantly more insulin (Mastrolia et al., 2017). Functional characterization shows that both male and female β-cells display a similar glucose-induced increase in the membrane potential under all stimulatory glucose levels (e.g. ≅10mM glucose RMPfemales= −64.7±5.8 mV; RMPmales= −63.1±3.6 mV) consistent with similar HVCC Ca2+ influx and KATP, Kv, BK and SK K+ effluxes. The glucose induced intracellular Ca2+ transients are also similar between sexes consistent with identical Ca2+-induced Ca2+-release and intracellular store Ca2+ content. However, females have a higher percentage of β-cells that respond to glucose stimulation with increased electrical activity (≅10 mM glucose 58% of male β-cells fire action potentials (AP) compared to 90% of female β-cells). Additionally, female β-cells displayed a higher frequency of glucose-induced AP-trains (≅10 mM, female β-cells 2.2±0.5 AP-trains/min, males 0.41±0.1, p<0.05). The increased electrical activity resulted in a significantly higher insulin release in females compared to males therefore lowering the risk of developing T2DM. Current experiments are on the way to elucidate the mechanism leading to the higher electrical activity in female β-cells. Support: FWF P31434, P36053, and DOC30-B30 to PT.

Sinoatrial node cells and tissue operate via stochastic resonance.

Sinoatrial node (SAN), the primary heart pacemaker, is a highly heterogeneous, complex cellular network. Recent imaging data indicate that synchronized action potentials (APs) that exit the SAN emerge from heterogeneous signals, including subthreshold, irregular signals. We tested the hypothesis that SAN cells and tissue operate via stochastic resonance, i.e. biological noise within and among SAN cells plays an important role in generation of cardiac impulses. We performed patch-clamp experiments in current-clamp mode in isolated single rabbit SAN cells. When uniformly random current noise of a few pA amplitude with a zero average was applied via the patch pipette, cells that had been firing APs rarely and irregularly began firing more frequently and rhythmically. Further, similar noise application to non-firing cells (dormant cells or artificially hyperpolarized cells) initiated AP firing. These results were supported by simulations of Maltsev-Lakatta numerical model of SAN cells. Further, simulations using a numerical model of heterogeneous SAN cellular network, in which the rate of Ca pumping and ICaL conductance varied, revealed that random current noise applied within cells also improved the tissue function. In scenarios at the edge of criticality, in which the cell network could not operate (99.84% of non-firing cells), almost all cells (97.12%) fired APs when noise was applied to cells within the network. Although artificial noise was applied in our experiments and simulations, similar noise is likely to be generated under physiological conditions by stochastic openings of ion channels (RyRs and L-type Ca-channels) within cells and by subthreshold signals from neighboring cells in SAN tissue. Our results support the idea that the SAN operates via stochastic resonance, thus shifting the paradigm of SAN operation from dictatorship and democracy theories, to a complex neuronal-type modus operandi, i.e. the SAN operates as “the heart's brain”.