Current Ih Research Articles

Spinal Nerve Axotomy: Effects on Ih In Vivo and HCNs in DRG Neurons

In vitro experiments performed on dissociated dorsal root ganglion (DRG) neurons suggest the involvement of the hyperpolarization-activated cation current (Ih) in enhancing neuronal excitability, potentially contributing to neuropathic pain. However, the more confirmative in vivo information about how nerve injury interacts with Ih is lacking. In this study, Ih was recorded in vivo using the dynamic single-electrode voltage clamp (dSEVC) technique on L5 DRG neurons of normal rats and those seven days after spinal nerve axotomy (SNA). Compared to normal rats, SNA unexpectedly inhibited the activity of Ih channels on A-fiber DRG neurons: (a) the Ih current magnitude, density, and conductance were consistently diminished; and (b) the Ih activation velocity was slowed and the voltage for Ih activation was hyperpolarized. The half-activation voltage (V0.5) exhibited a negative shift, and the time constant for Ih activation was prolonged across all test potentials, indicating the reduced availability of Ih after SNA. To further investigate the mechanisms of SNA on Ih, the underlying HCN channels and the correlated mRNA were quantified and compared. The mRNA expression level of HCN1-4 was uniformly enhanced after SNA, which might have contributed to the increased cytoplasmic HCN1 intensity observed in both medium- and large-sized DRG neurons. This finding contradicted the functional reduction of Ih after SNA. Surprisingly, the HCN labeling pattern was altered after SNA: the labeling area of HCN1 and HCN2 at the membranous ring region of the axotomized large neurons became significantly thinner or absent. We concluded that the diminished ring immunoreactivity for HCN1 and HCN2 correlated with a reduced availability of Ih channels, elucidating the observed decrease in Ih in axotomized A-fiber neurons.

Cross-pathway integration of cAMP signals through cGMP and calcium-regulated phosphodiesterases in mouse striatal cholinergic interneurons.

Acetylcholine plays a key role in striatal function. Firing properties of striatal cholinergic interneurons depend on intracellular cAMP through the regulation of Ih currents. Yet, the dynamics of cyclic nucleotide signalling in these neurons have remained elusive. We used highly selective FRET biosensors and pharmacological compounds to analyse the functional contribution of phosphodiesterases in striatal cholinergic interneurons in mouse brain slices. PDE1A, PDE3A and PDE4 appear as the main controllers of cAMP levels in striatal cholinergic interneurons. The calcium signal elicited through NMDA or metabotropic glutamate receptors activates PDE1A, which degrades both cAMP and cGMP. Interestingly, the nitric oxide/cGMP pathway amplifies cAMP signalling via PDE3A inhibition-a mechanism hitherto unexplored in a neuronal context. The expression pattern of specific PDE enzymes in striatal cholinergic interneurons, by integrating diverse intracellular pathways, can adjust cAMP responses bidirectionally. These properties eventually allow striatal cholinergic interneurons to dynamically regulate their overall activity and modulate acetylcholine release. Remarkably, this effect is the opposite of the cGMP-induced inhibition of cAMP signals involving PDE2A in striatal medium-sized spiny neurons, which provides important insights for the understanding of signal integration in the striatum.

Ih block reveals separation of timescales in pyloric rhythm response to temperature changes in Cancer borealis.

Motor systems operate over a range of frequencies and relative timing (phase). We studied the role of the hyperpolarization-activated inward current (Ih) in regulating these features in the pyloric rhythm of the stomatogastric ganglion (STG) of the crab, Cancer borealis, as temperature was altered from 11°C to 21°C. Under control conditions, rhythm frequency increased monotonically with temperature, while the phases of the pyloric dilator (PD), lateral pyloric (LP), and pyloric (PY) neurons remained constant. Blocking Ih with cesium (Cs+) phase advanced PD offset, LP onset, and LP offset at 11°C, and the latter two further advanced as temperature increased. In Cs+ the frequency increase with temperature diminished and the Q10 of the frequency dropped from ~1.75 to ~1.35. Unexpectedly in Cs+, the frequency dynamics became non-monotonic during temperature transitions; frequency initially dropped as temperature increased, then rose once temperature stabilized, creating a characteristic 'jag'. Interestingly, these jags persisted during temperature transitions in Cs+ when the pacemaker was isolated by picrotoxin, although the temperature-induced change in frequency recovered to control levels. Overall, these data suggest that Ih plays an important role in maintaining smooth transitory responses and persistent frequency increases by different mechanisms in the pyloric circuitry during temperature fluctuations.

Ih block reveals separation of timescales in pyloric rhythm response to temperature changes in Cancer borealis

Motor systems operate over a range of frequencies and relative timing (phase). We studied the role of the hyperpolarization-activated inward current (Ih) in regulating these features in the pyloric rhythm of the stomatogastric ganglion (STG) of the crab, Cancer borealis, as temperature was altered from 11°C to 21°C. Under control conditions, rhythm frequency increased monotonically with temperature, while the phases of the pyloric dilator (PD), lateral pyloric (LP), and pyloric (PY) neurons remained constant. Blocking Ih with cesium (Cs+) phase advanced PD offset, LP onset, and LP offset at 11°C, and the latter two further advanced as temperature increased. In Cs+ the frequency increase with temperature diminished and the Q10 of the frequency dropped from ~1.75 to ~1.35. Unexpectedly in Cs+, the frequency dynamics became non-monotonic during temperature transitions; frequency initially dropped as temperature increased, then rose once temperature stabilized, creating a characteristic ‘jag’. Interestingly, these jags persisted during temperature transitions in Cs+ when the pacemaker was isolated by picrotoxin, although the temperature-induced change in frequency recovered to control levels. Overall, these data suggest that Ih plays an important role in maintaining smooth transitory responses and persistent frequency increases by different mechanisms in the pyloric circuitry during temperature fluctuations.

Deregulation of Melatonin Receptors and Differential Modulation of After-Hyperpolarization and Ih Currents Using Melatonin Treatment Due to Amyloid-β-Induced Neurotoxicity in the Hippocampus.

Treatment with melatonin is routinely prescribed for its potent antioxidant and cognitive-promoting effects, nevertheless, it has yet to find neuromodulatory effects in normal and disease conditions. Therefore, to investigate its neuromodulatory mechanisms, melatonin was systemically administered over 10 consecutive days to both intracortical normal saline- and amyloid-β 1-42 (Aβ) peptide-injected rats. At the behavioral level, treatment with melatonin was associated with reduced efficacy in restoring Aβ-induced deficit in passive-avoidance memory. Whole-cell patch-clamp recordings from CA1 pyramidal neurons revealed that melatonin treatment reduced spontaneous and evoked intrinsic excitability in control rats while exerting a reduction of spontaneous, but not evoked activity, in the Aβ-injected group. Interestingly, treatment with melatonin enhances after-hyperpolarization in control, but not Aβ-injected rats. In contrast, our voltage-clamp study showed that Ih current is significantly enhanced by Aβ injection, and this effect is further strengthened by treatment with melatonin in Aβ-injected rats. Finally, we discovered that the transcription of melatonin receptors 1 (MT1) and 2 (MT2) is significantly upregulated in the hippocampi of Aβ-injected rats. Collectively, our study demonstrates that systemic treatment with melatonin has differential neuromodulation on CA1 neuronal excitability, at least in part, via differential effects on after-hyperpolarization and Ih currents due to Aβ-induced neurotoxicity.

Selective Vulnerability of GABAergic Inhibitory Interneurons to Bilirubin Neurotoxicity in the Neonatal Brain.

Hyperbilirubinemia (HB) is a key risk factor for hearing loss in neonates, particularly premature infants. Here, we report that bilirubin (BIL)-dependent cell death in the auditory brainstem of neonatal mice of both sexes is significantly attenuated by ZD7288, a blocker for hyperpolarization-activated cyclic nucleotide-gated (HCN) channel-mediated current (I h), or by genetic deletion of HCN1. GABAergic inhibitory interneurons predominantly express HCN1, on which BIL selectively acts to increase their intrinsic excitability and mortality by enhancing HCN1 activity and Ca2+-dependent membrane targeting. Chronic BIL elevation in neonatal mice in vivo increases the fraction of spontaneously active interneurons and their firing frequency, I h, and death, compromising audition at the young adult stage in HCN1+/+, but not in HCN1-/- genotype. We conclude that HB preferentially targets HCN1 to injure inhibitory interneurons, fueling a feedforward loop in which lessening inhibition cascades hyperexcitability, Ca2+ overload, neuronal death, and auditory impairments. These findings rationalize HCN1 as a potential target for managing HB encephalopathy.

Ih Block Reveals Separation of Timescales in Pyloric Rhythm Response to Temperature Changes in Cancer borealis.

Motor systems operate over a range of frequencies and relative timing (phase). We studied the contribution of the hyperpolarization-activated inward current (Ih) to frequency and phase in the pyloric rhythm of the stomatogastric ganglion (STG) of the crab, Cancer borealis as temperature was altered from 11°C to 21°C. Under control conditions, the frequency of the rhythm increased monotonically with temperature, while the phases of the pyloric dilator (PD), lateral pyloric (LP), and pyloric (PY) neurons remained constant. When we blocked Ih with cesium (Cs+) PD offset, LP onset, and LP offset were all phase advanced in Cs+ at 11°C, and the latter two further advanced as temperature increased. In Cs+ the steady state increase in pyloric frequency with temperature diminished and the Q10 of the pyloric frequency dropped from ~1.75 to ~1.35. Unexpectedly in Cs+, the frequency displayed non-monotonic dynamics during temperature transitions; the frequency initially dropped as temperature increased, then rose once temperature stabilized, creating a characteristic "jag". Interestingly, these jags were still present during temperature transitions in Cs+ when the pacemaker was isolated by picrotoxin, although the temperature-induced change in frequency recovered to control levels. Overall, these data suggest that Ih plays an important role in the ability of this circuit to produce smooth transitory responses and persistent frequency increases by different mechanisms during temperature fluctuations.

Muscarinic receptor activation preferentially inhibits rebound in vulnerable dopaminergic neurons.

Dopaminergic subpopulations of the substantia nigra pars compacta (SNc) differentially degenerate in Parkinson's disease and are characterized by unique electrophysiological properties. The vulnerable population expresses a T-type calcium channel-mediated afterdepolarization (ADP) and shows rebound activity upon release from inhibition, whereas the resilient population does not have an ADP and is slower to fire after hyperpolarization. This rebound activity can trigger dopamine release in the striatum, an important component of basal ganglia function. Using whole-cell patch clamp electrophysiology on ex vivo slices from adult mice of both sexes, we find that muscarinic activation with the non-selective muscarinic agonist Oxotremorine inhibits rebound activity more strongly in vulnerable vs resilient SNc neurons. Here, we show that this effect depends on the direct activation of muscarinic receptors on the SNc dopaminergic neurons. Through a series of pharmacological and transgenic knock-out experiments, we tested whether the muscarinic inhibition of rebound was mediated through the canonical rebound-related ion channels: T-type calcium channels, hyperpolarization-activated cation channels (HCN), and A-type potassium channels. We find that muscarinic receptor activation inhibits HCN-mediated current (Ih) in vulnerable SNc neurons, but that Ih activity is not necessary for the muscarinic inhibition of rebound activity. Similarly, we find that Oxotremorine inhibits rebound activity independently of T-type calcium channels and A-type potassium channels. Together these findings reveal new principles governing acetylcholine and dopamine interactions, showing that muscarinic receptors directly affect SNc rebound activity in the midbrain at the somatodendritic level and differentially modify information processing in distinct SNc subpopulations.

Hyperpolarization-activated currents drive neuronal activation sequences in sleep

Sequential neuronal patterns are believed to support information processing in the cortex, yet their origin is still a matter of debate. We report that neuronal activity in the mouse postsubiculum (PoSub), where a majority of neurons are modulated by the animal's head direction, was sequentially activated along the dorsoventral axis during sleep at the transition from hyperpolarized "DOWN" to activated "UP" states, while representing a stable direction. Computational modeling suggested that these dynamics could be attributed to a spatial gradient of hyperpolarization-activated currents (Ih), which we confirmed in exvivo slice experiments and corroborated in other cortical structures. These findings open up the possibility that varying amounts of Ih across cortical neurons could result in sequential neuronal patterns and that traveling activity upstream of the entorhinal-hippocampal circuit organizes large-scale neuronal activity supporting learning and memory during sleep.

Effective suppression of Ih and INa caused by capsazepine, known to be a blocker of TRPV1 receptor

A synthetic inhibitor of capsaicin-induced TRPV1 channel activation is called capsazepine (CPZ). In this study, we aimed to explore the effects of CPZ on hyperpolarization-activated cationic current (Ih) and voltage-gated Na + current (INa) in pituitary tumor (GH3) cells. Through patch-clamp recordings, we found that CPZ concentration-dependently inhibited Ih amplitude and slowed its activation time course. The IC50 and KD values were 3.1 and 3.16 μM, respectively. CPZ also shifted the steady-state activation curve of Ih towards a more hyperpolarized potential. However, there was no change in the gating charge of the curve. A modified Markovian model predicted the CPZ-induced decrease in the voltage-dependent hysteresis of Ih. CPZ suppressed INa in GH3 cells, without altering its activation or inactivation time course. Additionally, exposure to CPZ reduced spontaneous firing. These findings suggest that CPZ’s inhibitory effects on Ih and INa are direct and not dependent on vanilloid receptor binding. This could provide light on an unidentified ionic mechanism influencing the membrane excitability of neurons and endocrine or neuroendocrine cells in vivo.

Chronic alcohol consumption alters sex-dependent BNST neuron function in rhesus macaques

Repeated alcohol drinking contributes to a number of neuropsychiatric diseases, including alcohol use disorder and co-expressed anxiety and mood disorders. Women are more susceptible to the development and expression of these diseases with the same history of alcohol exposure as men, suggesting they may be more sensitive to alcohol-induced plasticity in limbic brain regions controlling alcohol drinking, stress responsivity, and reward processing, among other behaviors. Using a translational model of alcohol drinking in rhesus monkeys, we examined sex differences in the basal function and plasticity of neurons in the bed nucleus of the stria terminalis (BNST), a brain region in the extended amygdala shown to be a hub circuit node dysregulated in individuals with anxiety and alcohol use disorder. We performed slice electrophysiology recordings from BNST neurons in male and female monkeys following daily “open access” (22 h/day) to 4% ethanol and water for more than one year or control conditions. We found that BNST neurons from control females had reduced overall current density, hyperpolarization-activated depolarizing current (Ih), and inward rectification, as well as higher membrane resistance and greater synaptic glutamatergic release and excitatory drive, than those from control males, suggesting that female BNST neurons are more basally excited than those from males. Chronic alcohol drinking produced a shift in these measures in both sexes, decreasing current density, Ih, and inward rectification and increasing synaptic excitation. In addition, network activity-dependent synaptic inhibition was basally higher in BNST neurons of males than females, and alcohol exposure increased this in both sexes, a putative homeostatic mechanism to counter hyperexcitability. Altogether, these results suggest that the rhesus BNST is more basally excited in females than males and chronic alcohol drinking produces an overall increase in excitability and synaptic excitation. These results shed light on the mechanisms contributing to the female-biased susceptibility to neuropsychiatric diseases including co-expressed anxiety and alcohol use disorder.

Dexmedetomidine Prolongs Lidocaine Intravenous Regional Anesthesia in Rats by Blocking the Hyperpolarization-Activated Cation Current.

Intravenous regional anesthesia (IVRA) using lidocaine provides effective localized analgesia but its duration is limited. The mechanism by which dexmedetomidine enhances lidocaine IVRA is unclear but may involve modulation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Lidocaine IVRA with varying dexmedetomidine concentrations was performed in the tails of Sprague-Dawley rats. Tail-flick and tail-clamping tests assessed IVRA analgesia and anesthesia efficacy and duration. Contributions of α2 adrenergic receptors and HCN channels were evaluated by incorporating an α adrenergic receptor antagonist, the HCN channel inhibitor ZD7288, and the HCN channel agonist forskolin. Furthermore, whole-cell patch clamp electrophysiology quantified the effects of dexmedetomidine on HCN channels mediating hyperpolarization-activated cation current (Ih) in isolated dorsal root ganglion neurons. Dexmedetomidine dose-dependently extended lidocaine IVRA duration and analgesia, unaffected by α2 receptor blockade. The HCN channel inhibitor ZD7288 also prolonged lidocaine IVRA effects, while the HCN channel activator forskolin shortened effects. In dorsal root ganglion neurons, dexmedetomidine concentration-dependently inhibited Ih amplitude and shifted the voltage-dependence of HCN channel activation. Dexmedetomidine prolongs lidocaine IVRA duration by directly inhibiting HCN channel activity, independent of α2 adrenergic receptor activation. This HCN channel inhibition represents a novel mechanism underlying the anesthetic and analgesic adjuvant effects of dexmedetomidine in IVRA.

Electrophysiological Properties of Proprioception-Related Neurons in the Intermediate Thoracolumbar Spinal Cord.

Proprioception, the sense of limb and body position, is required to produce accurate and precise movements. Proprioceptive sensory neurons transmit muscle length and tension information to the spinal cord. The function of excitatory neurons in the intermediate spinal cord, which receive this proprioceptive information, remains poorly understood. Using genetic labeling strategies and patch-clamp techniques in acute spinal cord preparations in mice, we set out to uncover how two sets of spinal neurons, Clarke's column (CC) and Atoh1-lineage neurons, respond to electrical activity and how their inputs are organized. Both sets of neurons are located in close proximity in laminae V-VII of the thoracolumbar spinal cord and have been described to receive proprioceptive signals. We find that a majority of CC neurons have a tonic-firing type and express a distinctive hyperpolarization-activated current (Ih). Atoh1-lineage neurons, which cluster into two spatially distinct populations, are mostly a fading-firing type and display similar electrophysiological properties to each other, possibly due to their common developmental lineage. Finally, we find that CC neurons respond to stimulation of lumbar dorsal roots, consistent with prior knowledge that CC neurons receive hindlimb proprioceptive information. In contrast, using a combination of electrical stimulation, optogenetic stimulation, and transsynaptic rabies virus tracing, we find that Atoh1-lineage neurons receive heterogeneous, predominantly local thoracic inputs that include parvalbumin-lineage sensory afferents and local interneuron presynaptic inputs. Altogether, we find that CC and Atoh1-lineage neurons have distinct membrane properties and sensory input organization, representing different subcircuit modes of proprioceptive information processing.

Impaired amygdala astrocytic signaling worsens neuropathic pain-associated neuronal functions and behaviors.

Introduction: Pain is a clinically relevant health care issue with limited therapeutic options, creating the need for new and improved analgesic strategies. The amygdala is a limbic brain region critically involved in the regulation of emotional-affective components of pain and in pain modulation. The central nucleus of amygdala (CeA) serves major output functions and receives nociceptive information via the external lateral parabrachial nucleus (PB). While amygdala neuroplasticity has been linked causally to pain behaviors, non-neuronal pain mechanisms in this region remain to be explored. As an essential part of the neuroimmune system, astrocytes that represent about 40-50% of glia cells within the central nervous system, are required for physiological neuronal functions, but their role in the amygdala remains to be determined for pain conditions. In this study, we measured time-specific astrocyte activation in the CeA in a neuropathic pain model (spinal nerve ligation, SNL) and assessed the effects of astrocyte inhibition on amygdala neuroplasticity and pain-like behaviors in the pain condition. Methods and Results: Glial fibrillary acidic protein (GFAP, astrocytic marker) immunoreactivity and mRNA expression were increased at the chronic (4weeks post-SNL), but not acute (1week post-SNL), stage of neuropathic pain. In order to determine the contribution of astrocytes to amygdala pain-mechanisms, we used fluorocitric acid (FCA), a selective inhibitor of astrocyte metabolism. Whole-cell patch-clamp recordings were performed from neurons in the laterocapsular division of the CeA (CeLC) obtained from chronic neuropathic rats. Pre-incubation of brain slices with FCA (100µM, 1h), increased excitability through altered hyperpolarization-activated current (Ih) functions, without significantly affecting synaptic responses at the PB-CeLC synapse. Intra-CeA injection of FCA (100µM) had facilitatory effects on mechanical withdrawal thresholds (von Frey and paw pressure tests) and emotional-affective behaviors (evoked vocalizations), but not on facial grimace score and anxiety-like behaviors (open field test), in chronic neuropathic rats. Selective inhibition of astrocytes by FCA was confirmed with immunohistochemical analyses showing decreased astrocytic GFAP, but not NeuN, signal in the CeA. Discussion: Overall, these results suggest a complex modulation of amygdala pain functions by astrocytes and provide evidence for beneficial functions of astrocytes in CeA in chronic neuropathic pain.

Characterization of hyperpolarization-activated cyclic nucleotide-gated channels in oligodendrocytes.

Mature oligodendrocytes (OLG) are the myelin-forming cells of the central nervous system. Recent work has shown a dynamic role for these cells in the plasticity of neural circuits, leading to a renewed interest in voltage-sensitive currents in OLG. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and their respective current (Ih) were recently identified in mature OLG and shown to play a role in regulating myelin length. Here we provide a biochemical and electrophysiological characterization of HCN channels in cells of the oligodendrocyte lineage. We observed that mice with a nonsense mutation in the Hcn2 gene (Hcn2ap/ap) have less white matter than their wild type counterparts with fewer OLG and fewer oligodendrocyte progenitor cells (OPCs). Hcn2ap/ap mice have severe motor impairments, although these deficits were not observed in mice with HCN2 conditionally eliminated only in oligodendrocytes (Cnpcre/+; Hcn2F/F). However, Cnpcre/+; Hcn2F/F mice develop motor impairments more rapidly in response to experimental autoimmune encephalomyelitis (EAE). We conclude that HCN2 channels in OLG may play a role in regulating metabolism.

Dynamics of antiphase bursting modulated by the inhibitory synaptic and hyperpolarization-activated cation currents.

Antiphase bursting related to the rhythmic motor behavior exhibits complex dynamics modulated by the inhibitory synaptic current (Isyn), especially in the presence of the hyperpolarization-activated cation current (Ih). In the present paper, the dynamics of antiphase bursting modulated by the Ih and Isyn is studied in three aspects with a theoretical model. Firstly, the Isyn and the slow Ih with strong strength are the identified to be the necessary conditions for the antiphase bursting. The dependence of the antiphase bursting on the two currents is different for low (escape mode) and high (release mode) threshold voltages (Vth) of the inhibitory synapse. Secondly, more detailed co-regulations of the two currents to induce opposite changes of the bursting period are obtained. For the escape mode, increase of the Ih induces elevated membrane potential of the silence inhibited by a strong Isyn and shortened silence duration to go beyond Vth, resulting in reduced bursting period. For the release mode, increase of the Ih induces elevated tough value of the former part of the burst modulated by a nearly zero Isyn and lengthen burst duration to fall below Vth, resulting in prolonged bursting period. Finally, the fast-slow dynamics of the antiphase bursting are acquired. Using one-and two-parameter bifurcations of the fast subsystem of a single neuron, the burst of the antiphase bursting is related to the stable limit cycle, and the silence modulated by a strong Isyn to the stable equilibrium to a certain extent. The Ih mainly modulates the dynamics within the burst and quiescent state. Furthermore, with the fast subsystem of the coupled neurons, the silence is associated with the unstable equilibrium point. The results present theoretical explanations to the changes in the bursting period and fast-slow dynamics of the antiphase bursting modulated by the Isyn and Ih, which is helpful for understanding the antiphase bursting and modulating rhythmic motor patterns.

Dynamics of interaction between IH and IKLT currents to mediate double resonances of medial superior olive neurons related to sound localization.

Neurons in the medial superior olive (MSO) exhibit high frequency responses such as subthreshold resonance, which is helpful to sensitively detect a small difference in the arrival time of sounds between two ears for precise sound localization. Recently, except for the high frequency depolarization resonance mediated by a low threshold potassium (IKLT) current, a low frequency hyperpolarization resonance mediated by a hyperpolarization-activated cation (IH) current is observed in experiments on the MSO neurons, forming double resonances. The complex dynamics underlying double resonances are studied in an MSO neuron model in the present paper. Firstly, double resonances similar to the experimental observations are simulated as the resting membrane potential is between half-activation voltages of IH and IKLT currents, and stimulation current (IZAP) with large amplitude and exponentially increasing frequency is applied. Secondly, multiple effective factors to modulate double resonances are obtained. Especially, the decrease of time constant of IKLT current and increase of conductance of IH and IKLT currents can enhance the depolarization resonance frequency for precise sound localization. Last, different frequency responses of slow IH and fast IKLT currents in formation of the resonances are acquired. A middle phase difference between IZAP and IKLT currents appears at a high frequency, and the interaction between the positive part of IZAP and the negative IKLT current forms the depolarization resonance. Interaction between the negative part of IZAP and positive IH current with a middle phase difference results in hyperpolarization resonance at a low frequency. Furthermore, the phase difference between IZAP and resonance current can well explain the increase of depolarization resonance frequency modulated by the increase of conductance of IH or IKLT currents. The results present the dynamical and biophysical mechanisms for the double resonances mediated by two currents in the MSO neurons, which is helpful to enhance the depolarization resonance frequency for precise sound localization.

Inhibitory Perturbations of Fluvastatin on Afterhyperpolarization Current, Erg-mediated K+ Current, and Hyperpolarization-activated Cation Current in Both Pituitary GH3 Cells and Primary Embryonic Mouse Cortical Neurons.

Fluvastatin (FLV), the first synthetically derived 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, is a potent inhibitor of cholesterol biosynthesis. While its primary mechanism of action is to reduce cholesterol levels, there is some evidence suggesting that it may also have effects on K+ channels. However, the overall effects of fluvastatin on ionic currents are not yet well understood. The whole-cell clamp recordings were applied to evaluate the ionic currents and action potentials of cells. Here, we have demonstrated that FLV can effectively inhibit the amplitude of erg-mediated K+ current (IK(erg)) in pituitary tumor (GH3) cells, with an IC50 of approximately 3.2µM. In the presence of FLV, the midpoint in the activation curve of IK(erg) was distinctly shifted to a less negative potential by 10mV, with minimal modification of the gating charge. However, the magnitude of hyperpolarization-activated cation current (Ih) elicited by long-lasting membrane hyperpolarization was progressively decreased, with an IC50 value of 8.7µM, upon exposure to FLV. More interestingly, we also found that FLV (5µM) could regulate the action potential and afterhyperpolarization properties in primary embryonic mouse cortical neurons. Our study presents compelling evidence indicating that FLV has the potential to impact both the amplitude and gating of the ion channels IK(erg) and Ih. We also provide credible evidence suggesting that this drug has the potential to modify the properties of action potentials and the afterhyperpolarization current in electrically excitable cells. However, the assumption that these findings translate to similar in-vivo results remains unclear.

Ih current stabilizes excitability in rodent DRG neurons and reverses hyperexcitability in a nociceptive neuron model of inherited neuropathic pain.

We show here that hyperpolarization-activated current (Ih ) unexpectedly acts to inhibit the activity of dorsal root ganglion (DRG) neurons expressing WT Nav1.7, the largest inward current and primary driver of DRG neuronal firing, and hyperexcitable DRG neurons expressing a gain-of-function Nav1.7 mutation that causes inherited erythromelalgia (IEM), a human genetic model of neuropathic pain. In this study we created a kinetic model of Ih and used it, in combination with dynamic-clamp, to study Ih function in DRG neurons. We show, for the first time, that Ih increases rheobase and reduces the firing probability in small DRG neurons, and demonstrate that the amplitude of subthreshold oscillations is reduced by Ih . Our results show that Ih , due to slow gating, is not deactivated during action potentials (APs) and has a striking damping action, which reverses from depolarizing to hyperpolarizing, close to the threshold for AP generation. Moreover, we show that Ih reverses the hyperexcitability of DRG neurons expressing a gain-of-function Nav1.7 mutation that causes IEM. In the aggregate, our results show that Ih unexpectedly has strikingly different effects in DRG neurons as compared to previously- and well-studied cardiac cells. Within DRG neurons where Nav1.7 is present, Ih reduces depolarizing sodium current inflow due to enhancement of Nav1.7 channel fast inactivation and creates additional damping action by reversal of Ih direction from depolarizing to hyperpolarizing close to the threshold for AP generation. These actions of Ih limit the firing of DRG neurons expressing WT Nav1.7 and reverse the hyperexcitability of DRG neurons expressing a gain-of-function Nav1.7 mutation that causes IEM. KEY POINTS: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, the molecular determinants of hyperpolarization-activated current (Ih ) have been characterized as a 'pain pacemaker', and thus considered to be a potential molecular target for pain therapeutics. Dorsal root ganglion (DRG) neurons express Nav1.7, a channel that is not present in central neurons or cardiac tissue. Gain-of-function mutations (GOF) of Nav1.7 identified in inherited erythromelalgia (IEM), a human genetic model of neuropathic pain, produce DRG neuron hyperexcitability, which in turn produces severe pain. We found that Ih increases rheobase and reduces firing probability in small DRG neurons expressing WT Nav1.7, and demonstrate that the amplitude of subthreshold oscillations is reduced by Ih . We also demonstrate that Ih reverses the hyperexcitability of DRG neurons expressing a GOF Nav1.7 mutation (L858H) that causes IEM. Our results show that, in contrast to cardiac cells and CNS neurons, Ih acts to stabilize DRG neuron excitability and prevents excessive firing.

Assessing neuroprotective effects of diroximel fumarate and siponimod via modulation of pacemaker channels in an experimental model of remyelination

Cuprizone (CPZ)-induced alterations in axonal myelination are associated with a period of neuronal hyperexcitability and increased activity of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels in the thalamocortical (TC) system. Substances used for the treatment of multiple sclerosis (MS) have been shown to normalize neuronal excitability in CPZ-treated mice. Therefore, we aimed to examine the effects of diroximel fumarate (DRF) and the sphingosine 1-phospate receptor (S1PR) modulator siponimod on action potential firing and the inward current (Ih) carried by HCN ion channels in naive conditions and during different stages of de- and remyelination. Here, DRF application reduced Ih current density in ex vivo patch clamp recordings from TC neurons of the ventrobasal thalamic complex (VB), thereby counteracting the increase of Ih during early remyelination. Siponimod reduced Ih in VB neurons under control conditions but had no effect in neurons of the auditory cortex (AU). Furthermore, siponimod increased and decreased AP firing properties of neurons in VB and AU, respectively. Computational modeling revealed that both DRF and siponimod influenced thalamic bursting during early remyelination by delaying the onset and decreasing the interburst frequency. Thus, substances used in MS treatment normalize excitability in the TC system by influencing AP firing and Ih.