Hyperpolarization-activated Cation Research Articles

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.

The influence of hyperpolarization-activated cation current on conduction delay and failure of action potentials along axon related to abnormal functions

The influence of hyperpolarization-activated cation current on conduction delay and failure of action potentials along axon related to abnormal functions

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.

Chronic but not acute nicotine treatment ameliorates acute inflammation-induced working memory impairment by increasing CRTC1 and HCN2 in adult male mice.

Systemic inflammation in which lipopolysaccharide (LPS) is released into circulation can cause cognitive dysfunction and we have previously shown that LPS impaired working memory (WM) which refers to the ability to guide incoming behavior by retrieving recently acquired information. However, the mechanism is not very clear, and currently, there is no approved strategy to improve inflammation-induced WM deficit. Notably, epidemiological studies have demonstrated a lower occurrence rate of inflammatory-related diseases in smoking patients, suggesting that inflammation-induced WM impairment may be improved by nicotine treatment. Here, our object is to investigate the effect and potential mechanisms of acute and chronic nicotine treatment on LPS-produced WM deficiency. Delayed alternation T-maze task (DAT) was applied for evaluating WM which includes both the short-term information storage and the ability to correct errors in adult male mice. Immunofluorescence staining and immunoblotting were used for assessing the levels and distribution of CREB-regulated transcription coactivator 1 (CRTC1) and hyperpolarization-activated cation channels 2 (HCN2) in the medial prefrontal cortex (mPFC) and hippocampus. Quantitative PCR and ELISA were employed for analyzing the mRNA and protein levels of TNF-α and IL-1β. Our results revealed that administration of LPS (i.p.) at a dose of 0.5 mg/kg significantly produced WM impairment in the DAT task accompanied by an increase in IL-1β and TNF-α expression in the mPFC. Moreover, intra-mPFC infusion of IL-1Ra, an IL-1 antagonist, markedly alleviated LPS-induced WM deficiency. More important, chronic (2 weeks) but not acute nicotine (0.2 mg/kg, subcutaneous) treatment significantly alleviated LPS-induced WM deficiency by upregulating CRTC1 and HCN2. Of note, intra-mPFC infusion of HCN blocker ZD7288 produced significant WM deficiency. In summary, in this study, we show that chronic nicotine treatment ameliorates acute inflammation-induced working memory deficiency by increasing CRTC1 and HCN2 in adult male mice.

Missing Puzzle Pieces in Dementia Research: HCN Channels and Theta Oscillations.

Increasing evidence indicates a role of hyperpolarization activated cation (HCN) channels in controlling the resting membrane potential, pacemaker activity, memory formation, sleep, and arousal. Their disfunction may be associated with the development of epilepsy and age-related memory decline. Neuronal hyperexcitability involved in epileptogenesis and EEG desynchronization occur in the course of dementia in human Alzheimer's Disease (AD) and animal models, nevertheless the underlying ionic and cellular mechanisms of these effects are not well understood. Some suggest that theta rhythms involved in memory formation could be used as a marker of memory disturbances in the course of neurogenerative diseases, including AD. This review focusses on the interplay between hyperpolarization HCN channels, theta oscillations, memory formation and their role(s) in dementias, including AD. While individually, each of these factors have been linked to each other with strong supportive evidence, we hope here to expand this linkage to a more inclusive picture. Thus, HCN channels could provide a molecular target for developing new therapeutic agents for preventing and/or treating dementia.

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.

Cannabidiol Modulates M-Type K+ and Hyperpolarization-Activated Cation Currents.

Cannabidiol (CBD) is a naturally occurring compound found in the Cannabis plant that is known for its potential therapeutic effects. However, its impact on membrane ionic currents remains a topic of debate. This study aimed to investigate how CBD modifies various types of ionic currents in pituitary GH3 cells. Results showed that exposure to CBD led to a concentration-dependent decrease in M-type K+ currents (IK(M)), with an IC50 of 3.6 μM, and caused the quasi-steady-state activation curve of the current to shift to a more depolarized potential with no changes in the curve's steepness. The CBD-mediated block of IK(M) was not reversed by naloxone, suggesting that it was not mediated by opioid receptors. The IK(M) elicited by pulse-train stimulation was also decreased upon exposure to CBD. The magnitude of erg-mediated K+ currents was slightly reduced by adding CBD (10 μM), while the density of voltage-gated Na+ currents elicited by a short depolarizing pulse was not affected by it. Additionally, CBD decreased the magnitude of hyperpolarization-activated cation currents (Ih) with an IC50 of 3.3 μM, and the decrease was reversed by oxaliplatin. The quasi-steady-state activation curve of Ih was shifted in the leftward direction with no changes in the slope factor of the curve. CBD also diminished the strength of voltage-dependent hysteresis on Ih elicited by upright isosceles-triangular ramp voltage. Collectively, these findings suggest that CBD's modification of ionic currents presented herein is independent of cannabinoid or opioid receptors and may exert a significant impact on the functional activities of excitable cells occurring in vitro or in vivo.

Modulating Hyperpolarization-Activated Cation Currents through Small Molecule Perturbations: Magnitude and Gating Control.

The hyperpolarization-activated cation current (Ih) exhibits a slowly activating time course of the current (Ih) when the cell membrane is hyperpolarized for an extended duration. It is involved in generating electrical activity in various excitable cells. Numerous structurally distinct compounds or herbal drugs have the potential to impact both the magnitude and gating kinetics of this current. Brivaracetam, a chemical analog of levetiracetam known to be a ligand for synaptic vesicle protein 2A, could directly suppress the Ih magnitude. Carisbamate, an anticonvulsant agent, not only inhibited the Ih amplitude but also reduced the strength of voltage-dependent hysteresis (Hys(V)) associated with Ih. Cilobradine, similar to ivabradine, inhibited the amplitude of Ih; however, it also suppressed the amplitude of delayed-rectifier K+ currents. Dexmedetomidine, an agonist of α2-adrenergic receptor, exerted a depressant action on Ih in a concentration-dependent fashion. Suppression of Ih amplitude was observed when GAL-021, a breathing control modulator, was present at a concentration exceeding 30 μM. Lutein, one of the few xanthophyll carotenoids, was able to suppress the Ih amplitude as well as to depress Hys(V)'s strength of Ih. Pirfenidone, a pyridine derivative known to be an anti-fibrotic agent, depressed the Ih magnitude in a concentration- and voltage-dependent fashion. Tramadol, a synthetic centrally active analgesic, was shown to reduce the Ih magnitude, independent of its interaction with opioid receptors. Various herbal drugs, including ent-kaurane-type diterpenoids from Croton tonkinensis, Ganoderma triterpenoids, honokiol, and pterostilbene, demonstrated efficacy in reducing the magnitude of Ih. Conversely, oxaliplatin, a platinum-based chemotherapeutic compound, was observed to effectively increase the Ih amplitude. Collectively, the regulatory effects of these compounds or herbal drugs on cellular function can be partly attributed to their perturbations on Ih.

Hyperpolarization-activated cation channels shape the spiking frequency preference of human cortical layer 5 pyramidal neurons.

Discerning the contribution of specific ionic currents to complex neuronal dynamics is a difficult, but important, task. This challenge is exacerbated in the human setting, although the widely-characterized uniqueness of the human brain as compared to preclinical models necessitates the direct study of human neurons. Neuronal spiking frequency preference is of particular interest given its role in rhythm generation and signal transmission in cortical circuits. Here, we combine the frequency-dependent gain (FDG), a measure of spiking frequency preference, and novel in silico analyses to dissect the contributions of individual ionic currents to the suprathreshold features of human L5 neurons captured by the FDG. We confirm that a contemporary model of such a neuron, primarily constrained to capture subthreshold activity driven by the hyperpolarization-activated cyclic nucleotide gated (h-) current, replicates key features of the in vitro FDG both with and without h-current activity. With the model confirmed as a viable approximation of the biophysical features of interest, we applied new analysis techniques to quantify the activity of each modeled ionic current in the moments prior to spiking, revealing unique dynamics of the h-current. These findings motivated patch-clamp recordings in analogous rodent neurons to characterize their FDG, which confirmed that a biophysically-detailed model of these neurons captures key inter-species differences in the FDG. These differences are correlated with distinct contributions of the h-current to neuronal activity. Together, this interdisciplinary and multi-species study provides new insights directly relating the dynamics of the h-current to suprathreshold spiking frequency preference in human L5 neurons.Significance StatementUnderstanding the contributions of individual ionic currents to neuronal activity is vital, considering the established role of ion channel modifications in neuropsychiatric conditions. We combine in vitro characterization of the spiking frequency preference of human L5 cortical pyramidal neurons via the frequency-dependent gain (FDG) with new analyses of a biophysically-detailed computational model of such a neuron to delineate the connection between the dynamics of the hyperpolarization-activated cyclic nucleotide gated (h-) current prior to spiking and key properties of the FDG. By further determining that both these FDG properties and h-current dynamics are distinct in analogous rodent neurons, we provide convincing evidence for the key role of the h-current in the suprathreshold frequency preference of human L5 cortical neurons.

Concerted suppressive effects of carisbamate, an anti-epileptic alkyl-carbamate drug, on voltage-gated Na+ and hyperpolarization-activated cation currents.

Carisbamate (CRS, RWJ-333369) is a new anti-seizure medication. It remains unclear whether and how CRS can perturb the magnitude and/or gating kinetics of membrane ionic currents, despite a few reports demonstrating its ability to suppress voltage-gated Na+ currents. In this study, we observed a set of whole-cell current recordings and found that CRS effectively suppressed the voltage-gated Na+ (INa) and hyperpolarization-activated cation currents (Ih) intrinsically in electrically excitable cells (GH3 cells). The effective IC50 values of CRS for the differential suppression of transient (INa(T)) and late INa (INa(L)) were 56.4 and 11.4 μM, respectively. However, CRS strongly decreased the strength (i.e., Δarea) of the nonlinear window component of INa (INa(W)), which was activated by a short ascending ramp voltage (Vramp); the subsequent addition of deltamethrin (DLT, 10 μM) counteracted the ability of CRS (100 μM, continuous exposure) to suppress INa(W). CRS strikingly decreased the decay time constant of INa(T) evoked during pulse train stimulation; however, the addition of telmisartan (10 μM) effectively attenuated the CRS (30 μM, continuous exposure)-mediated decrease in the decay time constant of the current. During continued exposure to deltamethrin (10 μM), known to be a pyrethroid insecticide, the addition of CRS resulted in differential suppression of the amplitudes of INa(T) and INa(L). The amplitude of Ih activated by a 2-s membrane hyperpolarization was diminished by CRS in a concentration-dependent manner, with an IC50 value of 38 μM. For Ih, CRS altered the steady-state I-V relationship and attenuated the strength of voltage-dependent hysteresis (Hys(V)) activated by an inverted isosceles-triangular Vramp. Moreover, the addition of oxaliplatin effectively reversed the CRS-mediated suppression of Hys(V). The predicted docking interaction between CRS and with a model of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel or between CRS and the hNaV1.7 channel reflects the ability of CRS to bind to amino acid residues in HCN or hNaV1.7 channel via hydrogen bonds and hydrophobic interactions. These findings reveal the propensity of CRS to modify INa(T) and INa(L) differentially and to effectively suppress the magnitude of Ih. INa and Ih are thus potential targets of the actions of CRS in terms of modulating cellular excitability.

Strength-duration properties and excitability of motor and sensory axons across different target thresholds.

The present series of studies aimed to investigate the biophysical basis underlying differences in behavior between motor and sensory axons at different target response levels. In 24 healthy individuals, axonal excitability protocols measured strength-duration properties and latent addition across several axonal populations, with target amplitudes set at 10%, 20%, 40%, and 60%. Strength-duration time constants (SDTCs) were typically longer at lower target levels for both motor and sensory axons. Threshold change at 0.2 ms during assessment of latent addition, representing a persistent Na+ current (Nap), was higher in sensory axons. Passive membrane properties were not different across target levels. Significant relationships were evident between the threshold change at 0.2 ms and SDTC across all target levels for motor and sensory axons. These differences were explored using mathematical modeling of excitability data. With decreasing target size, as the internodal leak conductance increased in sensory axons, the Barrett-Barrett conductance decreased, whereas the hyperpolarization-activated cation current (Ih) channels became more depolarized. A similar pattern was observed in motor axons. As such, it was concluded that Nap was not responsible for the differences observed in SDTC between different target levels, although within specific target levels, Nap changes contributed to the variability of SDTC. This study provides a comprehensive assessment of Nap current, SDTC, and outlines key factors operating at different target levels in motor and sensory axons. Findings from the present study may point to the contributing factors of symptom development in human neuropathy.NEW & NOTEWORTHY This study provides a comprehensive assessment concerning the strength-duration behavior of motor and sensory axons at differing target levels of the compound nerve response. Strength-duration time constant was increased at lower target response levels particularly for sensory axons, whereas threshold change at 0.2 ms and passive membrane properties were not different. The results have established templates for axonal behavior in normal human axons, demonstrating altered adaptive responses, presumably secondary to different patterns of nerve activation.

An evolutionarily conserved pacemaker role for HCN ion channels in smooth muscle.

Although hyperpolarization-activated cation (HCN) ion channels are well established to underlie cardiac pacemaker activity, their role in smooth muscle organs remains controversial. HCN-expressing cells are localized to renal pelvic smooth muscle (RPSM) pacemaker tissues of the murine upper urinary tract and HCN channel conductance is required for peristalsis. To date, however, the Ih pacemaker current conducted by HCN channels has never been detected in these cells, raising questions on the identity of RPSM pacemakers. Indeed, the RPSM pacemaker mechanisms of the unique multicalyceal upper urinary tract exhibited by humans remains unknown. Here, we developed immunopanning purification protocols and demonstrate that 96% of isolated HCN+ cells exhibit Ih . Single-molecule STORM to whole-tissue imaging showed HCN+ cells express single HCN channels on their plasma membrane and integrate into the muscular syncytium. By contrast, PDGFR-α+ cells exhibiting the morphology of ICC gut pacemakers were shown to be vascular mural cells. Translational studies in the homologous human and porcine multicalyceal upper urinary tracts showed that contractions and pacemaker depolarizations originate in proximal calyceal RPSM. Critically, HCN+ cells were shown to integrate into calyceal RPSM pacemaker tissues, and HCN channel block abolished electrical pacemaker activity and peristalsis of the multicalyceal upper urinary tract. Cumulatively, these studies demonstrate that HCN ion channels play a broad, evolutionarily conserved pacemaker role in both cardiac and smooth muscle organs and have implications for channelopathies as putative aetiologies of smooth muscle disorders. KEY POINTS: Pacemakers trigger contractions of involuntary muscles. Hyperpolarization-activated cation (HCN) ion channels underpin cardiac pacemaker activity, but their role in smooth muscle organs remains controversial. Renal pelvic smooth muscle (RPSM) pacemakers trigger contractions that propel waste away from the kidney. HCN+ cells localize to murine RPSM pacemaker tissue and HCN channel conductance is required for peristalsis. The HCN (Ih ) current has never been detected in RPSM cells, raising doubt whether HCN+ cells are bona fide pacemakers. Moreover, the pacemaker mechanisms of the unique multicalyceal RPSM of higher order mammals remains unknown. In total, 97% of purified HCN+ RPSM cells exhibit Ih . HCN+ cells integrate into the RPSM musculature, and pacemaker tissue peristalsis is dependent on HCN channels. Translational studies in human and swine demonstrate HCN channels are conserved in the multicalyceal RPSM and that HCN channels underlie pacemaker activity that drives peristalsis. These studies provide insight into putative channelopathies that can underlie smooth muscle dysfunction.

Reduced oriens-lacunosum/moleculare cell model identifies biophysical current balances for in vivo theta frequency spiking resonance.

Conductance-based models have played an important role in the development of modern neuroscience. These mathematical models are powerful "tools" that enable theoretical explorations in experimentally untenable situations, and can lead to the development of novel hypotheses and predictions. With advances in cell imaging and computational power, multi-compartment models with morphological accuracy are becoming common practice. However, as more biological details are added, they make extensive explorations and analyses more challenging largely due to their huge computational expense. Here, we focus on oriens-lacunosum/moleculare (OLM) cell models. OLM cells can contribute to functionally relevant theta rhythms in the hippocampus by virtue of their ability to express spiking resonance at theta frequencies, but what characteristics underlie this is far from clear. We converted a previously developed detailed multi-compartment OLM cell model into a reduced single compartment model that retained biophysical fidelity with its underlying ion currents. We showed that the reduced OLM cell model can capture complex output that includes spiking resonance in in vivo-like scenarios as previously obtained with the multi-compartment model. Using the reduced model, we were able to greatly expand our in vivo-like scenarios. Applying spike-triggered average analyses, we were able to to determine that it is a combination of hyperpolarization-activated cation and muscarinic type potassium currents that specifically allow OLM cells to exhibit spiking resonance at theta frequencies. Further, we developed a robust Kalman Filtering (KF) method to estimate parameters of the reduced model in real-time. We showed that it may be possible to directly estimate conductance parameters from experiments since this KF method can reliably extract parameter values from model voltage recordings. Overall, our work showcases how the contribution of cellular biophysical current details could be determined and assessed for spiking resonance. As well, our work shows that it may be possible to directly extract these parameters from current clamp voltage recordings.

HCN-Channel-Dependent Hyperexcitability of the Layer V Pyramidal Neurons in IL-mPFC Contributes to Fentanyl-Induced Hyperalgesia in Male Rats.

Opioids are often first-line analgesics in pain therapy. However, prolonged use of opioids causes paradoxical pain, termed "opioid-induced hyperalgesia (OIH)." The infralimbic medial prefrontal cortex (IL-mPFC) has been suggested to be critical in inflammatory and neuropathic pain processing through its dynamic output from layer V pyramidal neurons. Whether OIH condition induces excitability changes of these output neurons and what mechanisms underlie these changes remains elusive. Here, with combination of patch-clamp recording, immunohistochemistry, as well as optogenetics, we revealed that IL-mPFC layer V pyramidal neurons exhibited hyperexcitability together with higher input resistance. In line with this, optogenetic and chemogenetic activation of these neurons aggravates behavioral hyperalgesia in male OIH rats. Inhibition of these neurons alleviates hyperalgesia in male OIH rats but exerts an opposite effect in male control rats. Electrophysiological analysis of hyperpolarization-activated cation current (Ih) demonstrated that decreased Ih is a prerequisite for the hyperexcitability of IL-mPFC output neurons. This decreased Ih was accompanied by a decrease in HCN1, but not HCN2, immunolabeling, in these neurons. In contrast, the application of HCN channel blocker increased the hyperalgesia threshold of male OIH rats. Consequently, we identified an HCN-channel-dependent hyperexcitability of IL-mPFC output neurons, which governs the development and maintenance of OIH in male rats.

Bi-directional modulation of hyperpolarization-activated cation currents (Ih) by ethanol in rat hippocampal CA3 pyramidal neurons

It is widely acknowledged that ethanol (EtOH) can alter many neuronal functions, including synaptic signaling, firing discharge, and membrane excitability, through its interaction with multiple membrane proteins and intracellular pathways. Previous work has demonstrated that EtOH enhances the firing rate of hippocampal GABAergic interneurons and thus the presynaptic GABA release at CA1 and CA3 inhibitory synapses through a positive modulation of the hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels. Activation of HCN channels produce an inward current, commonly called Ih, which plays an essential role in generating/regulating specific neuronal activities in GABAergic interneurons and principal glutamatergic pyramidal neurons such as those in the CA3 subregion. Since the direct effect of EtOH on HCN channels expressed in CA3 pyramidal neurons was not thoroughly elucidated, we investigated the possible interaction between EtOH and HCN channels and the impact on excitability and postsynaptic integration of these neurons. Patch-clamp recordings were performed in single CA3 pyramidal neurons from acute male rat coronal hippocampal slices. Our results show that EtOH modulates HCN-mediated Ih in a concentration-dependent and bi-directional manner, with a positive modulation at lower (20 mM) and an inhibitory action at higher (60–80 mM) concentrations. The modulation of Ih by EtOH was mimicked by forskolin, antagonized by different drugs that selectively interfere with the AC/cAMP/PKA intracellular pathway, as well as by the selective HCN inhibitor ZD7288. Altogether, these data further support the evidence that HCN channels may represent an important molecular target through which EtOH may regulate neuronal activity.

The Modulation of Ubiquinone, a Lipid Antioxidant, on Neuronal Voltage-Gated Sodium Current.

Ubiquinone, composed of a 1,4-benzoquinone and naturally produced in the body, actively participates in the mitochondrial redox reaction and functions as an endogenous lipid antioxidant, protecting against peroxidation in the pituitary-dependent hormonal system. However, the questions of if and how ubiquinone directly affects neuronal ionic currents remain largely unsettled. We investigated its effects on ionic currents in pituitary neurons (GH3 and MMQ cells) with the aid of patch-clamp technology. Ubiquinone decreased the peak amplitude of the voltage-gated Na+ current (INa) with a slowing of the inactivation rate. Neither menadione nor superoxide dismutase modified the ubiquinone-induced INa inhibition. In response to an isosceles-triangular ramp pulse, the persistent INa (INa(P)) at high- and low- threshold potentials occurred concurrently with a figure-eight hysteresis loop. With ubiquinone, the INa(P) increased with no change in the intersection voltage, and the magnitude of the voltage-dependent hysteresis of the current was enhanced. Ubiquinone was ineffective in modifying the gating of hyperpolarization-activated cation currents. In MMQ lactotrophs, ubiquinone effectively decreased the amplitude of the INa and the current inactivation rate. In sum, the effects of ubiquinone demonstrated herein occur upstream of its effects on mitochondrial redox processes, involved in its modulation of sodium channels and neuronal excitability.

Characterization of Inhibitory Capability on Hyperpolarization-Activated Cation Current Caused by Lutein (β,ε-Carotene-3,3'-Diol), a Dietary Xanthophyll Carotenoid.

Lutein (β,ε-carotene-3,3′-diol), a xanthophyll carotenoid, is found in high concentrations in the macula of the human retina. It has been recognized to exert potential effectiveness in antioxidative and anti-inflammatory properties. However, whether and how its modifications on varying types of plasmalemmal ionic currents occur in electrically excitable cells remain incompletely answered. The current hypothesis is that lutein produces any direct adjustments on ionic currents (e.g., hyperpolarization-activated cation current, Ih [or funny current, If]). In the present study, GH3-cell exposure to lutein resulted in a time-, state- and concentration-dependent reduction in Ih amplitude with an IC50 value of 4.1 μM. There was a hyperpolarizing shift along the voltage axis in the steady-state activation curve of Ih in the presence of this compound, despite being void of changes in the gating charge of the curve. Under continued exposure to lutein (3 μM), further addition of oxaliplatin (10 μM) or ivabradine (3 μM) could be effective at either reversing or further decreasing lutein-induced suppression of hyperpolarization-evoked Ih, respectively. The voltage-dependent anti-clockwise hysteresis of Ih responding to long-lasting inverted isosceles-triangular ramp concentration-dependently became diminished by adding this compound. However, the addition of 10 μM lutein caused a mild but significant suppression in the amplitude of erg-mediated or A-type K+ currents. Under current-clamp potential recordings, the sag potential evoked by long-lasting hyperpolarizing current stimulus was reduced under cell exposure to lutein. Altogether, findings from the current observations enabled us to reflect that during cell exposure to lutein used at pharmacologically achievable concentrations, lutein-perturbed inhibition of Ih would be an ionic mechanism underlying its changes in membrane excitability.

Adenosine Downregulates the Activities of Glutamatergic Neurons in the Paraventricular Hypothalamic Nucleus Required for Sleep.

Adenosine is an endogenous substance that regulates sleep homeostasis. It plays an important role in sleep induction under physiological condition. So far, the neural mechanisms underlying sleep-promoting effects of adenosine are not completely clear. Recent studies have shown that glutamatergic neurons in the paraventricular hypothalamic nucleus (PVH) play an important role in wakefulness. Using whole-cell patch-clamp, we found that adenosine can inhibit glutamatergic neurons in PVH. This inhibition is mainly achieved by activating adenosine type 1 receptors, thereby reducing hyperpolarization-activated cyclic nucleotide-gated cation channels. By recording electroencephalogram (EEG) and electromyography (EMG), it was found that local administration of adenosine type 1 receptor blocker in PVH could significantly reduce the NREM sleep. On the contrary, if adenosine was given, it could increase the NREM sleep. These results suggest that adenosine can promote sleep by reducing the excitability of PVH neurons. This findings reveal a novel mechanism of adenosine regulating sleep homeostasis.

Role of the Dopamine Subtype 1 Receptor (D1R) Modulation of the Ih Current in Rhythmic Spinal Mammalian Motor Networks

Restless Legs Syndrome (RLS) is a chronic sensorimotor disorder that has been recently associated with decreased expression of inhibitory Gi‐coupled adenosine A1 receptors (A1Rs), leading to increased dopamine release (hyperdopaminergic signaling) within the central nervous system. Previous studies have shown that A1Rs specifically inhibit the Gs‐coupled dopamine D1 receptor (D1R) subtype through a functional A1R‐D1R heteromer receptor complex. Furthermore, D1Rs control spinal motor neuron (MN) excitability by increasing cyclic adenosine monophosphate (cAMP) concentration and opening hyperpolarization‐activated cyclic nucleotide‐gated non‐selective cation channel (HCN). This finding raises the possibility of increased HCN activity underlying some of the motor dysfunctions associated with periodic leg movements (PLMS) during sleep in RLS. Thus, this study aims to elucidate the importance of the D1 receptor subtype on modulating the hyperpolarization‐activated cation current (Ih) in spinal mammalian motor networks. I hypothesized that blocking Ih in the presence of a D1R agonist (enhanced dopaminergic modulation) would slow locomotor activity and disrupt MN bursting stability. Neonatal (P0‐P5; n = 7) mouse spinal cord preparations were used to measure MN bursting activity patterns including amplitude, duration, and cycle period of extracellular ventral root (L2 and L5) recordings via suction electrodes. Treatment conditions include serotonin (9‐15 μM 5‐HT) and N‐methyl‐D‐aspartate (6 μM NMDA) with and without a D1R agonist (10 μM SKF38393) to assess the effect of D1R on rhythmic bursting. Subsequently, an Ih blocker (1 μM ZD7288) was added to observe the importance of Ih in modulating MN burst activity. For both L2 and L5 roots, blocking the Ih current led to increases in burst amplitude, duration, and cycle period in respect to the control 5‐HT/NMDA condition. Our results propose that D1Rs play a significant role in modulating Ih by increasing cAMP levels and opening HCN1 channels at a more depolarized membrane potential. Future experiments involve: 1) using TM5 transmembrane disrupting peptide to disrupt A1R‐D1R heteromers in spinal MNs (and use TM7 as a negative control) to confirm if dopamine modulation of Ih activity is linked to the A1R‐D1R heteromer complexes and 2) applying adenyl cyclase activators and blockers to verify that the modulation of the Ih current is through the D1R‐mediated Gs second messenger pathway.