Enhanced Action Potential Firing Research Articles

A SCN8A variant associated with severe early onset epilepsy and developmental delay: Loss- or gain-of-function?

SCN8A, encoding the voltage-gated sodium channel subunit NaV1.6, has been associated with a wide spectrum of neuropsychiatric disorders. Missense variants in SCN8A which increase the channel activity can cause a severe developmental and epileptic encephalopathy (DEE). One DEE variant (p.(Arg223Gly)) was described to cause a predominant loss-of-function (LOF) mechanism when expressed in neuroblastoma cells, which is not consistent with the genotype-phenotype correlations in this gene. To resolve this discrepancy and understand the pathophysiological mechanism of this variant, we performed comprehensive electrophysiological studies in both neuroblastoma cells and primary hippocampal neuronal cultures. Although we also found that p.(Arg223Gly) significantly decreased Na+ current density and enhanced fast inactivation compared to the wild type (WT) channel in transfected neuroblastoma cells (both LOF mechanisms), it also caused a strong hyperpolarizing shift of steady-state activation and accelerated the recovery from fast inactivation (both gain-of-function (GOF) mechanisms). In cultured neurons transfected with mutant vs. WT NaV1.6 channels, we found more depolarized resting membrane potentials and a decreased rheobase leading to enhanced action potential firing. We conclude that SCN8A p.(Arg223Gly) leads to a net GOF resulting in neuronal hyperexcitability and a higher firing rate, fitting with the central role of GOF mechanisms in DEE.

Modulation of Vagal Sensory Neurons via High Mobility Group Box-1 and Receptor for Advanced Glycation End Products: Implications for Respiratory Viral Infections

Vagal sensory neurons contribute to the symptoms and pathogenesis of inflammatory pulmonary diseases through processes that involve changes to their morphological and functional characteristics. The alarmin high mobility group box-1 (HMGB1) is an early mediator of pulmonary inflammation and can have actions on neurons in a range of inflammatory settings. We hypothesized that HMGB1 can regulate the growth and function of vagal sensory neurons and we set out to investigate this and the mechanisms involved. Culturing primary vagal sensory neurons from wildtype mice in the presence of HMGB1 significantly increased neurite outgrowth, while acute application of HMGB1 to isolated neurons under patch clamp electrophysiological investigation produced inward currents and enhanced action potential firing. Transcriptional analyses revealed the expression of the cognate HMGB1 receptors, Receptor for Advanced Glycation End products (RAGE) and Toll-like Receptor 4 (TLR4), in subsets of vagal sensory neurons. HMGB1-evoked growth and electrophysiological responses were significantly reduced in primary vagal sensory neurons harvested from RAGE deficient mice and completely absent in neurons from RAGE/TLR4 double deficient mice. Immunohistochemical analysis of vagal sensory neurons collected from mice after intranasal infection with murine pneumovirus or influenza A virus (IAV), or after intratracheal administration with the viral mimetic PolyI:C, revealed a significant increase in nuclear-to-cytoplasm translocation of HMGB1 compared to mock-inoculated mice. Neurons cultured from virus infected wildtype mice displayed a significant increase in neurite outgrowth, which was not observed for neurons from virus infected RAGE or RAGE/TLR4 deficient mice. These data suggest that HMGB1 can enhance vagal sensory neuron growth and excitability, acting primarily via sensory neuron RAGE. Activation of the HMGB1-RAGE axis in vagal sensory neurons could be an important mechanism leading to vagal hyperinnervation and hypersensitivity in chronic pulmonary disease.

Ion-channel degeneracy: Multiple ion channels heterogeneously regulate intrinsic physiology of rat hippocampal granule cells.

Degeneracy, the ability of multiple structural components to elicit the same characteristic functional properties, constitutes an elegant mechanism for achieving biological robustness. In this study, we sought electrophysiological signatures for the expression of ion‐channel degeneracy in the emergence of intrinsic properties of rat hippocampal granule cells. We measured the impact of four different ion‐channel subtypes—hyperpolarization‐activated cyclic‐nucleotide‐gated (HCN), barium‐sensitive inward rectifier potassium (Kir), tertiapin‐Q‐sensitive inward rectifier potassium, and persistent sodium (NaP) channels—on 21 functional measurements employing pharmacological agents, and report electrophysiological data on two characteristic signatures for the expression of ion‐channel degeneracy in granule cells. First, the blockade of a specific ion‐channel subtype altered several, but not all, functional measurements. Furthermore, any given functional measurement was altered by the blockade of many, but not all, ion‐channel subtypes. Second, the impact of blocking each ion‐channel subtype manifested neuron‐to‐neuron variability in the quantum of changes in the electrophysiological measurements. Specifically, we found that blocking HCN or Ba‐sensitive Kir channels enhanced action potential firing rate, but blockade of NaP channels reduced firing rate of granule cells. Subthreshold measures of granule cell intrinsic excitability (input resistance, temporal summation, and impedance amplitude) were enhanced by blockade of HCN or Ba‐sensitive Kir channels, but were not significantly altered by NaP channel blockade. We confirmed that the HCN and Ba‐sensitive Kir channels independently altered sub‐ and suprathreshold properties of granule cells through sequential application of pharmacological agents that blocked these channels. Finally, we found that none of the sub‐ or suprathreshold measurements of granule cells were significantly altered upon treatment with tertiapin‐Q. Together, the heterogeneous many‐to‐many mapping between ion channels and single‐neuron intrinsic properties emphasizes the need to account for ion‐channel degeneracy in cellular‐ and network‐scale physiology.

Chronic Nicotine Exposure Alters the Neurophysiology of Habenulo-Interpeduncular Circuitry.

Antagonism of nicotinic acetylcholine receptors (nAChRs) in the medial habenula (MHb) or interpeduncular nucleus (IPN) triggers withdrawal-like behaviors in mice chronically exposed to nicotine, implying that nicotine dependence involves the sensitization of nicotinic signaling. Identification of receptor and/or neurophysiological mechanisms underlying this sensitization is important, as it could promote novel therapeutic strategies to reduce tobacco use. Using an approach involving photoactivatable nicotine, we previously demonstrated that chronic nicotine (cNIC) potently enhances nAChR function in dendrites of MHb neurons. However, whether cNIC modulates downstream components of the habenulo-interpeduncular (Hb-IP) circuit is unknown. In this study, cNIC-mediated changes to Hb-IP nAChR function were examined in mouse (male and female) brain slices using molecular, electrophysiological, and optical techniques. cNIC enhanced action potential firing and modified spike waveform characteristics in MHb neurons. Nicotine uncaging revealed nAChR functional enhancement by cNIC on proximal axonal membranes. Similarly, nAChR-driven glutamate release from MHb axons was enhanced by cNIC. In IPN, the target structure of MHb axons, neuronal morphology, and nAChR expression is complex, with stronger nAChR function in the rostral subnucleus [rostral IPN (IPR)]. As in MHb, cNIC induced strong upregulation of nAChR function in IPN neurons. This, coupled with cNIC-enhanced nicotine-stimulated glutamate release, was associated with stronger depolarization responses to brief (1 ms) nicotine uncaging adjacent to IPR neurons. Together, these results indicate that chronic exposure to nicotine dramatically alters nicotinic cholinergic signaling and cell excitability in Hb-IP circuits, a key pathway involved in nicotine dependence.SIGNIFICANCE STATEMENT This study uncovers several neuropharmacological alterations following chronic exposure to nicotine in a key brain circuit involved in nicotine dependence. These results suggest that smokers or regular users of electronic nicotine delivery systems (i.e., "e-cigarettes") likely undergo sensitization of cholinergic circuitry in the Hb-IP system. Reducing the activity of Hb-IP nAChRs, either volitionally during smoking cessation or inadvertently via receptor desensitization during nicotine intake, may be a key trigger of withdrawal in nicotine dependence. Escalation of nicotine intake in smokers, or tolerance, may involve stimulation of these sensitized cholinergic pathways. Smoking cessation therapeutics are only marginally effective, and by identifying cellular/receptor mechanisms of nicotine dependence, our results take a step toward improved therapeutic approaches for this disorder.

Cell-Type-Specific D1 Dopamine Receptor Modulation of Projection Neurons and Interneurons in the Prefrontal Cortex.

Dopamine modulation in the prefrontal cortex (PFC) mediates diverse effects on neuronal physiology and function, but the expression of dopamine receptors at subpopulations of projection neurons and interneurons remains unresolved. Here, we examine D1 receptor expression and modulation at specific cell types and layers in the mouse prelimbic PFC. We first show that D1 receptors are enriched in pyramidal cells in both layers 5 and 6, and that these cells project to intratelencephalic targets including contralateral cortex, striatum, and claustrum rather than to extratelencephalic structures. We then find that D1 receptors are also present in interneurons and enriched in superficial layer VIP-positive (VIP+) interneurons that coexpresses calretinin but absent from parvalbumin-positive (PV+) and somatostatin-positive (SOM+) interneurons. Finally, we determine that D1 receptors strongly and selectively enhance action potential firing in only a subset of these corticocortical neurons and VIP+ interneurons. Our findings define several novel subpopulations of D1+ neurons, highlighting how modulation via D1 receptors can influence both excitatory and disinhibitory microcircuits in the PFC.

Induction of thermal and mechanical hypersensitivity by parathyroid hormone-related peptide through upregulation of TRPV1 function and trafficking.

The neurobiological mechanisms underlying chronic pain associated with cancers are not well understood. It has been hypothesized that factors specifically elevated in the tumor microenvironment sensitize adjacent nociceptive afferents. We show that parathyroid hormone-related peptide (PTHrP), which is found at elevated levels in the tumor microenvironment of advanced breast and prostate cancers, is a critical modulator of sensory neurons. Intraplantar injection of PTHrP led to the development of thermal and mechanical hypersensitivity in both male and female mice, which were absent in mice lacking functional transient receptor potential vanilloid-1 (TRPV1). The PTHrP treatment of cultured mouse sensory neurons enhanced action potential firing, and increased TRPV1 activation, which was dependent on protein kinase C (PKC) activity. Parathyroid hormone-related peptide induced robust potentiation of TRPV1 activation and enhancement of neuronal firing at mild acidic pH that is relevant to acidic tumor microenvironment. We also observed an increase in plasma membrane TRPV1 protein levels after exposure to PTHrP, leading to upregulation in the proportion of TRPV1-responsive neurons, which was dependent on the activity of PKC and Src kinases. Furthermore, co-injection of PKC or Src inhibitors attenuated PTHrP-induced thermal but not mechanical hypersensitivity. Altogether, our results suggest that PTHrP and mild acidic conditions could induce constitutive pathological activation of sensory neurons through upregulation of TRPV1 function and trafficking, which could serve as a mechanism for peripheral sensitization of nociceptive afferents in the tumor microenvironment.

Dynamic-clamp analysis of wild-type human Nav1.7 and erythromelalgia mutant channel L858H

The link between sodium channel Nav1.7 and pain has been strengthened by identification of gain-of-function mutations in patients with inherited erythromelalgia (IEM), a genetic model of neuropathic pain in humans. A firm mechanistic link to nociceptor dysfunction has been precluded because assessments of the effect of the mutations on nociceptor function have thus far depended on electrophysiological recordings from dorsal root ganglia (DRG) neurons transfected with wild-type (WT) or mutant Nav1.7 channels, which do not permit accurate calibration of the level of Nav1.7 channel expression. Here, we report an analysis of the function of WT Nav1.7 and IEM L858H mutation within small DRG neurons using dynamic-clamp. We describe the functional relationship between current threshold for action potential generation and the level of WT Nav1.7 conductance in primary nociceptive neurons and demonstrate the basis for hyperexcitability at physiologically relevant levels of L858H channel conductance. We demonstrate that the L858H mutation, when modeled using dynamic-clamp at physiological levels within DRG neurons, produces a dramatically enhanced persistent current, resulting in 27-fold amplification of net sodium influx during subthreshold depolarizations and even greater amplification during interspike intervals, which provide a mechanistic basis for reduced current threshold and enhanced action potential firing probability. These results show, for the first time, a linear correlation between the level of Nav1.7 conductance and current threshold in DRG neurons. Our observations demonstrate changes in sodium influx that provide a mechanistic link between the altered biophysical properties of a mutant Nav1.7 channel and nociceptor hyperexcitability underlying the pain phenotype in IEM.

MCH and apomorphine in combination enhance action potential firing of nucleus accumbens shell neuronsin vitro

The MCH and dopamine receptor systems have been shown to modulate a number of behaviors related to reward processing, addiction, and neuropsychiatric conditions such as schizophrenia and depression. In addition, MCH and dopamine receptors can interact in a positive manner, for example in the expression of cocaine self-administration. A recent report (Chung et al., 2011a) showed that the DA1/DA2 dopamine receptor activator apomorphine suppresses pre-pulse inhibition, a preclinical model for some aspects of schizophrenia. Importantly, MCH can enhance the effects of lower doses of apomorphine, suggesting that co-modulation of dopamine and MCH receptors might alleviate some symptoms of schizophrenia with a lower dose of dopamine receptor modulator and thus fewer potential side effects. Here, we investigated whether MCH and apomorphine could enhance action potential firing in vitro in the nucleus accumbens shell (NAshell), a region which has previously been shown to mediate some behavioral effects of MCH. Using whole-cell patch-clamp electrophysiology, we found that MCH, which has no effect on firing on its own, was able to increase NAshell firing when combined with a subthreshold dose of apomorphine. Further, this MCH/apomorphine increase in firing was prevented by an antagonist of either a DA1 or a DA2 receptor, suggesting that apomorphine acts through both receptor types to enhance NAshell firing. The MCH/apomorphine-mediated firing increase was also prevented by an MCH receptor antagonist or a PKA inhibitor. Taken together, our results suggest that MCH can interact with lower doses of apomorphine to enhance NAshell firing, and thus that MCH and apomorphine might interact in vivo within the NAshell to suppress pre-pulse inhibition.

Nucleotides control the excitability of sensory neurons via two P2Y receptors and a bifurcated signaling cascade

Nucleotides contribute to the sensation of acute and chronic pain, but it remained enigmatic which G protein-coupled nucleotide (P2Y) receptors and associated signaling cascades are involved. To resolve this issue, nucleotides were applied to dorsal root ganglion neurons under current- and voltage-clamp. Adenosine triphosphate (ATP), adenosine diphosphate (ADP), and uridine triphosphate (UTP), but not uridine diphosphate (UDP), depolarized the neurons and enhanced action potential firing in response to current injections. The P2Y2 receptor preferring agonist 2-thio-UTP was equipotent to UTP in eliciting these effects. The selective P2Y1 receptor antagonist MRS2179 largely attenuated the excitatory effects of ADP, but left those of 2-thio-UTP unaltered. Thus, the excitatory effects of the nucleotides were mediated by 2 different P2Y receptors, P2Y1 and P2Y2. Activation of each of these 2 receptors by either ADP or 2-thio-UTP inhibited currents through KV7 channels, on one hand, and facilitated currents through TRPV1 channels, on the other hand. Both effects were abolished by inhibitors of phospholipase C or Ca2+-ATPase and by chelation of intracellular Ca2+. The facilitation of TRPV1, but not the inhibition KV7 channels, was prevented by a protein kinase C inhibitor. Simultaneous blockage of KV7 channels and of TRPV1 channels prevented nucleotide-induced membrane depolarization and action potential firing. Thus, P2Y1 and P2Y2 receptors mediate an excitation of dorsal root ganglion neurons by nucleotides through the inhibition of KV7 channels and the facilitation of TRPV1 channels via a common bifurcated signaling pathway relying on an increase in intracellular Ca2+ and an activation of protein kinase C, respectively.

Cellular Correlates of Enhanced Anxiety Caused by Acute Treatment with the Selective Serotonin Reuptake Inhibitor Fluoxetine in Rats

Selective serotonin reuptake inhibitors (SSRIs) are used extensively in the treatment of depression and anxiety disorders. The therapeutic benefits of SSRIs typically require several weeks of continuous treatment. Intriguingly, according to clinical reports, symptoms of anxiety may actually increase during the early stages of treatment although more prolonged treatment alleviates affective symptoms. Consistent with earlier studies that have used animal models to capture this paradoxical effect of SSRIs, we find that rats exhibit enhanced anxiety-like behavior on the elevated plus-maze 1 h after a single injection of the SSRI fluoxetine. Next we investigated the potential neural substrates underlying the acute anxiogenic effects by analyzing the morphological and physiological impact of acute fluoxetine treatment on principal neurons of the basolateral amygdala (BLA), a brain area that plays a pivotal role in fear and anxiety. Although earlier studies have shown that behavioral or genetic perturbations that are anxiogenic for rodents also increase dendritic spine density in the BLA, we find that a single injection of fluoxetine does not cause spinogenesis on proximal apical dendritic segments on BLA principal neurons an hour later. However, at the same time point when a single dose of fluoxetine caused enhanced anxiety, it also enhanced action potential firing in BLA neurons in ex vivo slices. Consistent with this finding, in vitro bath application of fluoxetine caused higher spiking frequency and this increase in excitability was correlated with an increase in the input resistance of these neurons. Our results suggest that enhanced excitability of amygdala neurons may contribute to the increase in anxiety-like behavior observed following acute fluoxetine treatment.

Hyperpolarization-activated currents control the excitability of principal neurons in the basolateral amygdala

Anxiety is thought to be influenced by neuronal excitability in basolateral nucleus of the amygdala (BLA). However, molecules that are critical for regulating excitability of BLA neurons are yet to be determined. In the present study, we have examined whether hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels, which mediate the depolarizing cation current, can control the neuronal excitability. HCN channel-like activity appeared to be detected in BLA principal neurons. ZD7288, a specific blocker for HCN channels, increased the input resistance of membrane, hyperpolarized resting membrane potential, and enhanced action potential firing in BLA principal neurons. The blockade of HCN channels facilitated temporal summation of repetitively evoked excitatory postsynaptic potentials, suggesting that suppression of HCN channel activity in principal neurons can accelerate the propagation of synaptic responses onto the axon hillock. Thus, our findings have laid foundation for studies to reveal how HCN channel activity in BLA principal neurons regulates anxiety in vivo.

Phasic cholinergic signaling in the hippocampus: Functional homology with the neocortex?

Acetylcholine (ACh) acts as a neurotransmitter in both the hippocampus and neocortex to facilitate learning, memory, and cognitive function. Here we show that transient muscarinic ACh receptor (mAChR) activation inhibits action potential generation in CA1, but not in CA3, pyramidal neurons via activation of an SK-type calcium-activated potassium conductance. Hyperpolarizing responses generated by focal ACh application near the somata of CA1 pyramidal neurons were blocked by atropine or the M1-like mAChR antagonist pirenzepine, but not by the M2-like mAChR antagonist methoctramine. Inhibitory cholinergic responses required intracellular calcium signaling, as evidenced by their sensitivity to depletion of internal calcium stores or internal calcium chelation. Cholinergic inhibition did not require GABAergic synaptic transmission, but was blocked by apamin, an SK channel antagonist. In contrast to inhibitory effects in CA1 neurons, ACh was primarily depolarizing, and enhanced action potential firing in CA3 pyramidal neurons. These results, when combined with recent data in neocortical neurons, suggest a functional homology in phasic cholinergic signaling in the hippocampus and neocortex whereby ACh preferentially inhibits those neurons in the lower cortical layers (CA1 and layer 5 neurons) that provide the majority of extracortical efferent projections.

Dual-Level Afferent Control of Growth Hormone-Releasing Hormone (GHRH) Neurons in GHRH–Green Fluorescent Protein Transgenic Mice

The organization of the peptidergic neurons of the hypothalamic arcuate nucleus is not fully understood. These include growth hormone-releasing hormone (GHRH) neurons involved in growth and metabolism. We studied identified GHRH neurons of GHRH-green fluorescent protein transgenic mice using patch-clamp methods and focused on gender differences, which govern the physiological patterns of GHRH release. Both the spontaneous firing rates and the intrinsic properties of GHRH neurons were similar in males and females, although higher glutamatergic currents were noticed in females. Surprisingly, marked gender differences in GHRH neuronal activity were observed in response to the muscarinic agonist carbachol (CCh). In females, CCh enhanced action potential firing in all GHRH neurons. In males, CCh enhanced action potential firing in two-thirds of GHRH neurons, whereas it decreased firing in the remainders. M1 agonist McN-A343 (10 microM) mimicked, and M1 antagonist pirenzepine (3 microM) blocked the effects of CCh. In both genders, CCh did not change the intrinsic properties of GHRH neurons, although it strongly increased the frequency of glutamatergic currents, in the presence or absence of tetrodotoxin. In males only, CCh enhanced the frequency of GABAergic currents, and this modulation was antagonized by tetrodotoxin. Thus, the muscarinic regulation involved differential control of afferent inputs at short and long distances in male and female mice. The dual-level control could be a mechanism whereby the selective modulation of the GHRH system (short-distance control) is adjusted to the integrated regulation of arcuate nucleus activity (long-distance control).

Ca2+ Influx, But Not Ca2+ Release From Internal Stores, Is Required for the PACAP-Induced Increase in Excitability in Guinea Pig Intracardiac Neurons

Mechanisms modulating the pituitary adenylate cyclase activating polypeptide (PACAP)-induced increase in excitability have been studied using dissociated guinea pig intrinsic cardiac neurons and intact ganglion preparations. Measurements of intracellular calcium (Ca2+) with the fluorescent Ca2+ indicator dye fluo-3 indicated that neither PACAP nor vasoactive intestinal polypeptide (VIP) at either 100 nM or 1 microM produced a discernible elevation of intracellular Ca2+ in dissociated intracardiac neurons. For neurons in ganglion whole mount preparations kept in control bath solution, local application of PACAP significantly increased excitability, as indicated by the number of action potentials generated by long depolarizing current pulses. However, in a Ca2+ -deficient solution in which external Ca2+ was replaced by Mg2+ or when cells were bathed in control solution containing 200 microM Cd2+, PACAP did not enhance action potential firing. In contrast, in a Ca2+ -deficient solution with Ca2+ replaced by strontium (Sr2+), PACAP increased excitability. PACAP increased excitability in cells treated with a combination of 20 microM ryanodine and 10 mM caffeine to interrupt release of Ca2+ from internal stores. Experiments using fluo-3 showed that ryanodine/caffeine pretreatment eliminated subsequent caffeine-induced Ca2+ release from intracellular stores, whereas exposure to the Ca2+ -deficient solution did not. In dissociated intracardiac neurons voltage clamped with the perforated patch recording technique, 100 nM PACAP decreased the voltage-dependent barium current (IBa). These results show that, in the guinea pig intracardiac neurons, the PACAP-induced increase in excitability apparently requires Ca2+ influx through Cd2+ -sensitive calcium permeable channels other than voltage-dependent Ca2+ channels, but not Ca2+ release from internal stores.

VPAC Receptor Modulation of Neuroexcitability in Intracardiac Neurons: DEPENDENCE ON INTRACELLULAR CALCIUM MOBILIZATION AND SYNERGISTIC ENHANCEMENT BY PAC1 RECEPTOR ACTIVATION

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) have been found within mammalian intracardiac ganglia, but the cellular effects of these neuropeptides remain poorly understood. Fluorometric calcium imaging and whole cell patch clamp recordings were used to examine the effects of PACAP and VIP on [Ca2+]i and neuroexcitability, respectively, in intracardiac neurons of neonatal rats. PACAP and VIP evoked rapid increases in [Ca2+]i that exhibited both transient and sustained components. Pharmacological experiments using PAC1 and VPAC receptor-selective antagonists demonstrated that the elevations in [Ca2+]i result from the activation of VPAC receptors. The transient increases in [Ca2+]i were shown to be the product of Ca2+ mobilization from caffeine/ryanodine-sensitive intracellular stores and were not due to inositol 1,4,5-trisphosphate-mediated calcium release. In contrast, the sustained [Ca2+]i elevations were dependent on extracellular Ca2+ and were blocked by the transient receptor channel antagonist, 2-aminoethoxydiphenyl borate, which suggests that they are due to Ca2+ entry via store-operated channels. In addition to elevating [Ca2+]i, both PACAP and VIP depolarized intracardiac neurons, and PACAP was further shown to augment action potential firing in these cells. Depolarization of intracardiac neurons by the neuropeptides was dependent on activation of VPAC receptors and the concomitant increases in [Ca2+]i. Although activation of PAC1 receptors alone had no direct effects on neuroexcitability, PAC1 receptor stimulation potentiated the VPAC receptor-induced depolarizations. Furthermore, enhanced action potential firing was only observed upon concurrent stimulation of PAC1 and VPAC receptors, which indicates that these receptors act synergistically to enhance neuroexcitability in intracardiac neurons.