Hyperpolarization-activated Cation Research Articles

Firing properties of Renshaw cells defined by Chrna2 are modulated by hyperpolarizing and small conductance ion currents Ih and ISK.

Renshaw cells in the spinal cord ventral horn regulate motoneuron output through recurrent inhibition. Renshaw cells can be identified in vitro using anatomical and cellular criteria; however, their functional role in locomotion remains poorly defined because of the difficulty of functionally isolating Renshaw cells from surrounding motor circuits. Here we aimed to investigate whether the cholinergic nicotinic receptor alpha2 (Chrna2) can be used to identify Renshaw cells (RCs(α2)) in the mouse spinal cord. Immunohistochemistry and electrophysiological characterization of passive and active RCs(α2) properties confirmed that neurons genetically marked by the Chrna2-Cre mouse line together with a fluorescent reporter mouse line are Renshaw cells. Whole-cell patch-clamp recordings revealed that RCs(α2) constitute an electrophysiologically stereotyped population with a resting membrane potential of -50.5 ± 0.4 mV and an input resistance of 233.1 ± 11 MΩ. We identified a ZD7288-sensitive hyperpolarization-activated cation current (Ih) in all RCs(α2), contributing to membrane repolarization but not to the resting membrane potential in neonatal mice. Additionally, we found RCs(α2) to express small calcium-activated potassium currents (I(SK)) that, when blocked by apamin, resulted in a complete attenuation of the afterhyperpolarisation potential, increasing cellular firing frequency. We conclude that RCs(α2) can be genetically targeted through their selective Chrna2 expression and that they display currents known to modulate rebound excitation and firing frequency. The genetic identification of Renshaw cells and their electrophysiological profile is required for genetic and pharmacological manipulation as well as computational simulations with the aim to understand their functional role.

Hyperpolarization-activated cation current Ih influences synaptic integration properties in substantia nigra dopamine neurons

Event Abstract Back to Event Hyperpolarization-activated cation current Ih influences synaptic integration properties in substantia nigra dopamine neurons Dominique Engel1* and Vincent Seutin2 1 FNRS, GIGA-Neurosciences, Belgium 2 Université de Liège, GIGA-Neuroscience, Belgium The hyperpolarization-activated cation current Ih is expressed in many pyramidal neurons and in some interneurons. Ih contributes to neuronal signaling via the modulation of passive and active membrane properties, intrinsic resonance, synaptic integration and synaptic plasticity. One of the most characteristic functions of Ih is to influence synaptic integration by inducing a location independence of postsynaptic potentials (EPSPs) waveform and EPSP temporal summation. Herewith, EPSPs arriving at the soma have a similar time course, regardless of their origin. In pyramidal neurons, this effect of Ih is correlated to a specific subcellular distribution of the channel. In the substantia nigra, dopamine (DA) neurons are implicated in the control of movement. Their degeneration is related to motor disturbances as they are described in Parkinson’s disease. Interestingly, the axon of DA neurons generally emerges from a dendrite dividing the dendritic compartment of these neurons in axon- and non-axon bearing dendrites. This morphology contrasts with the typical arrangement of subcellular compartments found in neurons. DA neurons are also well known to display a prominent sag in response to long hyperpolarizing current injections, consecutive to slowly activating h-channels. However, the subcellular distribution of the channel and its role in synaptic integration is unknown for DA neurons. We performed cell-attached patch-clamp recordings along the somatodendritic domain of DA neurons and observed a high Ih current density in the axon-bearing dendrites in a membrane region close to the axon origin. Simultaneous whole-cell somatic and dendritic recordings revealed that Ih reduces EPSP integral and temporal summation. Keywords: Dopamine Neuron, Substantia Nigra, Dendrites, integration properties, hyperpolarization-activated inwardly rectifying cation conductance, Ih Conference: 11th National Congress of the Belgian Society for Neuroscience, Mons, Belgium, 22 May - 22 May, 2015. Presentation Type: Poster presentation Topic: Neuroscience Citation: Engel D and Seutin V (2015). Hyperpolarization-activated cation current Ih influences synaptic integration properties in substantia nigra dopamine neurons. Front. Neurosci. Conference Abstract: 11th National Congress of the Belgian Society for Neuroscience. doi: 10.3389/conf.fnins.2015.89.00016 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 05 May 2015; Published Online: 05 May 2015. * Correspondence: Dr. Dominique Engel, FNRS, GIGA-Neurosciences, Liege, 4000, Belgium, dominique.engel@uliege.be Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Dominique Engel Vincent Seutin Google Dominique Engel Vincent Seutin Google Scholar Dominique Engel Vincent Seutin PubMed Dominique Engel Vincent Seutin Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Inhibition of Hyperpolarization-Activated Cation Current in Medium-Sized DRG Neurons Contributed to the Antiallodynic Effect of Methylcobalamin in the Rat of a Chronic Compression of the DRG.

Recently several lines of evidence demonstrated that methylcobalamin (MeCbl) might have potential analgesic effect in experimental and clinical studies. However, it was reported that MeCbl had no effect on treating lumbar spinal stenosis induced pain. Thus, the effects of short-term and long-term administration of MeCbl were examined in the chronic compression of dorsal root ganglion (CCD) model. We found that mechanical allodynia was significantly inhibited by a continuous application of high dose and a single treatment of a super high dose of MeCbl. Little is known about mechanisms underlying the analgesia of MeCbl. We examined the effect of MeCbl on the spontaneous activity (SA), the excitability, and hyperpolarization-activated nonselective cation ion current in compressed medium-sized dorsal root ganglion (DRG) neurons using extracellular single fiber recording in vivo and whole-cell patch clamp in vitro. We found that MeCbl significantly inhibited the SA of A-type sensory neurons in a dose-dependent manner and inhibited the excitability of medium-sized DRG neurons. In addition, MeCbl also decreased I h current density in injured medium-sized DRG neurons. Our results proved that MeCbl might exert an analgesic effect through the inhibition I h current and then might inhibit the hyperexcitability of primary sensory neurons under neuropathic pain state.

Ionic mechanisms underlying the negative chronotropic action of propofol on sinoatrial node automaticity in guinea pig heart.

Propofol is a widely used intravenous anaesthetic agent, but has undesirable cardiac side effects, including bradyarrhythmia and its severe form asystole. This study examined the ionic and cellular mechanisms underlying propofol-induced bradycardia. Sinoatrial node cells, isolated from guinea pig hearts, were current- and voltage-clamped to record action potentials and major ionic currents involved in their spontaneous activity, such as the hyperpolarization-activated cation current (If ), T-type and L-type Ca(2+) currents (ICa,T and ICa,L , respectively) and the rapidly and slowly activating delayed rectifier K(+) currents (IKr and IK s , respectively). ECGs were recorded from Langendorff-perfused, isolated guinea pig hearts. Propofol (≥5 μM) reversibly decreased the firing rate of spontaneous action potentials and their diastolic depolarization rate. Propofol impaired If activation by shifting the voltage-dependent activation to more hyperpolarized potentials (≥1 μM), slowing the activation kinetics (≥3 μM) and decreasing the maximal conductance (≥10 μM). Propofol decreased ICa,T (≥3 μM) and ICa,L (≥1 μM). Propofol suppressed IKs (≥3 μM), but had a minimal effect on IKr . Furthermore, propofol (≥5 μM) decreased heart rates in Langendorff-perfused hearts. The sinoatrial node cell model reasonably well reproduced the negative chronotropic action of propofol. Micromolar concentrations of propofol suppressed the slow diastolic depolarization and firing rate of sinoatrial node action potentials by impairing If activation and reducing ICa,T , ICa,L and IKs . These observations suggest that the direct inhibitory effect of propofol on sinoatrial node automaticity, mediated via multiple channel inhibition, underlies the propofol-induced bradycardia observed in clinical settings.

Cocaine increases dopaminergic neuron and motor activity via midbrain α1 adrenergic signaling.

Cocaine reinforcement is mediated by increased extracellular dopamine levels in the forebrain. This neurochemical effect was thought to require inhibition of dopamine reuptake, but cocaine is still reinforcing even in the absence of the dopamine transporter. Here, we demonstrate that the rapid elevation in dopamine levels and motor activity elicited by cocaine involves α1 receptor activation within the ventral midbrain. Activation of α1 receptors increases dopaminergic neuron burst firing by decreasing the calcium-activated potassium channel current (SK), as well as elevates dopaminergic neuron pacemaker firing through modulation of both SK and the hyperpolarization-activated cation currents (Ih). Furthermore, we found that cocaine increases both the pacemaker and burst-firing frequency of rat ventral-midbrain dopaminergic neurons through an α1 adrenergic receptor-dependent mechanism within the ventral tegmental area and substantia nigra pars compacta. These results demonstrate the mechanism underlying the critical role of α1 adrenergic receptors in the regulation of dopamine neurotransmission and behavior by cocaine.

Loss of mitochondrial fission depletes axonal mitochondria in midbrain dopamine neurons.

Disruptions in mitochondrial dynamics may contribute to the selective degeneration of dopamine (DA) neurons in Parkinson's disease (PD). However, little is known about the normal functions of mitochondrial dynamics in these neurons, especially in axons where degeneration begins, and this makes it difficult to understand the disease process. To study one aspect of mitochondrial dynamics-mitochondrial fission-in mouse DA neurons, we deleted the central fission protein dynamin-related protein 1 (Drp1). Drp1 loss rapidly eliminates the DA terminals in the caudate-putamen and causes cell bodies in the midbrain to degenerate and lose α-synuclein. Without Drp1, mitochondrial mass dramatically decreases, especially in axons, where the mitochondrial movement becomes uncoordinated. However, in the ventral tegmental area (VTA), a subset of midbrain DA neurons characterized by small hyperpolarization-activated cation currents (Ih) is spared, despite near complete loss of their axonal mitochondria. Drp1 is thus critical for targeting mitochondria to the nerve terminal, and a disruption in mitochondrial fission can contribute to the preferential death of nigrostriatal DA neurons.

Differential effects of static and dynamic inputs on neuronal excitability.

The intrinsic excitability of neurons is known to be dynamically regulated by activity-dependent plasticity and homeostatic mechanisms. Such processes are commonly analyzed in the context of input-output functions that describe how neurons fire in response to constant levels of current. However, it is not well understood how changes of excitability as observed under static inputs translate to the function of the same neurons in their natural synaptic environment. Here we performed a computational study and hybrid experiments on rat bed nucleus of stria terminalis neurons to compare the two scenarios. The inward rectifying Kir current (IKir) and the hyperpolarization-activated cation current (Ih) were found to be considerably more effective in regulating the firing under synaptic inputs than under static stimuli. This prediction was experimentally confirmed by dynamic-clamp insertion of a synthetic inwardly rectifying Kir current into the biological neurons. At the same time, ionic currents that activate with depolarization were more effective regulating the firing under static inputs. When two intrinsic currents are concurrently altered such as those under homeostatic regulation, the effects in firing responses under static vs. dynamic inputs can be even more contrasting. Our results show that plastic or homeostatic changes of intrinsic membrane currents can shape the current step responses of neurons and their firing under synaptic inputs in a differential manner.

A detailed, conductance-based computer model of intrinsic sensory neurons of the gastrointestinal tract.

Intrinsic sensory neurons (ISNs) of the enteric nervous system respond to stimuli such as muscle tension, muscle length, distortion of the mucosa, and the chemical content in the lumen. ISNs form recurrent networks that probably drive many intestinal motor patterns and reflexes. ISNs express a large number of voltage- and calcium-gated ion channels, some of which are modified by inflammation or repeated physiological stimuli, but how interactions between different ionic currents in ISNs produce both normal and pathological behaviors in the intestine remains unclear. We constructed a model of ISNs including voltage-gated sodium and potassium channels, N-type calcium channels, big conductance calcium-dependent potassium (BK) channels, calcium-dependent nonspecific cation channels (NSCa), intermediate conductance calcium-dependent potassium (IK) channels, hyperpolarization-activated cation (Ih) channels, and internal calcium dynamics. The model was based on data from the literature and our electrophysiological studies. The model reproduced responses to short or long depolarizing current pulses and responses to long hyperpolarizing current pulses. Sensitivity analysis showed that Ih, IK, NSCa, and BK have the largest influence on the number of action potentials observed during prolonged depolarizations. The model also predicts that changes to the voltage of activation for Ih have a large influence on excitability, but changes to the time constant of activation for Ih have a minor effect. Our model identifies how interactions between different iconic currents influence the excitability of ISNs and highlights an important role for Ih in enteric neuroplasticity resulting from disease.

Entorhinal cortex stellate cell synchronization

Theta-frequency oscillations (8-12 Hz) of large groups of synaptically coupled cells are commonly seen throughout the mammalian brain. The theta oscillations of entorhinal cortex grid cells and hippocampal place cells have received special attention recently due to their role they apparently play in the encoding of positional information. In a previous study [1], we found that biologically realistic computational models of two or three synaptically coupled entorhinal cortex stellate cells [2] generally synchronize their oscillations much more quickly from an initially random phase configuration if their intrinsic firing frequencies are near the theta range (with the absolute minima generally at about 15-20 Hz) than at significantly either higher or lower ranges. However, our model looked only at identical neurons with the same intrinsic frequencies and lacking any channel noise. Here we present results from further studies in which realistic channel noise and frequency heterogeneity are included. We found that channel noise could either increase or decrease the time for coupled stellate cells to synchronize by a significant amount, depending on which channel the noise was applied to. We also found that the synchronization time appears to be largely independent of the underlying cell parameters that determine the intrinsic frequencies (the persistent sodium and hyperpolarization-activated cation conductances) and depend only on the frequencies themselves, potentially simplifying the process of parameter sweeps. We found evidence that synchronization is favored for a mean firing frequency of 18 Hz even when the individual intrinsic frequencies of the neurons differ by up to about 0.5 Hz (see Figure ​Figure1).1). Finally, we found evidence of a bifurcation in the stability of synchronization states of two coupled identical stellate cells, with in-phase synchronization suddenly switching to out-of-phase synchronization as the synaptic coupling strength is increased, suggesting a complex picture of entorhinal cortex synchronization patterns similar to previous studies of Hodgkin-Huxley neurons [3]. Figure 1 Long-term synchronization of two coupled entorhinal cortex stellate cell neurons with slightly different intrinsic firing frequencies. The mean of their intrinsic frequencies is plotted against a parameter (“Delta Coll 1”) relating the ...

Direct Negative Chronotropic Action of Desflurane on Sinoatrial Node Pacemaker Activity in the Guinea Pig Heart

Desflurane inhalation is associated with sympathetic activation and concomitant increase in heart rate in humans and experimental animals. There is, however, little information concerning the direct effects of desflurane on electrical activity of sinoatrial node pacemaker cells that determines the intrinsic heart rate. Whole-cell patch-clamp experiments were conducted on guinea pig sinoatrial node pacemaker cells to record spontaneous action potentials and ionic currents contributing to sinoatrial node automaticity, namely, hyperpolarization-activated cation current (If), T-type and L-type Ca currents (ICa,T and ICa,L, respectively), Na/Ca exchange current (INCX), and rapidly and slowly activating delayed rectifier K currents (IKr and IKs, respectively). Electrocardiograms were recorded from ex vivo Langendorff-perfused hearts and in vivo hearts. Desflurane at 6 and 12% decreased spontaneous firing rate of sinoatrial node action potentials by 15.9% (n = 11) and 27.6% (n = 10), respectively, which was associated with 20.4% and 42.5% reductions in diastolic depolarization rate, respectively. Desflurane inhibited If, ICa,T, ICa,L, INCX, and IKs but had little effect on IKr. The negative chronotropic action of desflurane was reasonably well reproduced in sinoatrial node computer model. Desflurane reduced the heart rate in Langendorff-perfused hearts. High concentration (12%) of desflurane inhalation was associated with transient tachycardia, which was totally abolished by pretreatment with the β-adrenergic blocker propranolol. Desflurane has a direct negative chronotropic action on sinoatrial node pacemaking activity, which is mediated by its inhibitory action on multiple ionic currents. This direct inhibitory action of desflurane on sinoatrial node automaticity seems to be counteracted by sympathetic activation associated with desflurane inhalation in vivo.

P853: Nerve excitability change by chronic administration of methylglyoxal: implication for diabetic neuropathy

Question: The methylglyoxal (MGO) pathway is an offshoot of glycolysis, which converts glucose into MGO. Frequently increased in diabetic patients, MGO is highly toxic, resulting in activation of apoptosis and mitochondrial damage. Recently, MGO was found to be a causative agent for neuropathic pain. Because abnormal nerve excitability is frequently present in peripheral neuropathy and neuropathic pain, MGO can alter overall nerve excitability. The objective of the present study was to observe the chronic effects of MGO on peripheral nerve excitability in vivo. Methods: Eight adult male mice were examined. Sensory nerve multiple excitability testing (QTRAC) was performed from a tail nerve under inhalational anesthesia. The parameters were compared between at baseline and 1 week after intraperitoneal injection of MGX (0.005 ml/10g body weight) nine times (3 times weekly for 3 weeks). Results: The tested animals were all survived. The excitability parameters were similar in strength-duration time constant and threshold changes by long depolarizing current (threshold electrotonus: TE). The threshold changes by long hyperpolarizing current (TE) became smaller by MGO; (1) TEh(peak) by -70% conditioning current: -203.8±7.5 at baseline to 162.3±9.1 post-injection, P=0.003; (2) TEh(90-100ms): -94.3±4.9 at baseline to -71.7±5.5 post-injection, P=0.006. The recovery cycle showed smaller refractoriness at 2ms by MGO: 23.5±3.0 at baseline to 7.9±4.8 post-injection, P=0.01. The overall nerve excitability assessed by strength-response curves was slightly reduced after injection. Conclusions: Chronic administration of MGO changed axonal excitability in vivo. It is likely that MGO facilitated hyperpolarization-activated cation current (Ih). The upregulation of Ih may be responsible for abnormal excitability and sensory symptoms of diabetic neuropathy.

Subthreshold delta‐frequency resonance in thalamic reticular neurons

The thalamic reticular nucleus (nRt) is an assembly of GABAergic projection neurons that participate in the generation of brain rhythms during synchronous sleep and absence epilepsy. NRt cells receive inhibitory and excitatory synaptic inputs, and are endowed with an intricate set of intrinsic conductances. However, little is known about how intrinsic and synaptic properties interact to generate rhythmic discharges in these neurons. In order to better understand this interaction, I studied the subthreshold responses of nRt cells to time-varying inputs. Patch-clamp recordings were performed in acute slices of rat thalamus (postnatal days 12-21). Sinusoidal current waveforms of linearly changing frequencies were injected into the soma, and the resulting voltage oscillations were recorded. At the resting membrane potential, the impedance profile showed a characteristic resonance at 1.7Hz. The relative strength of the resonance was 1.2, and increased with membrane hyperpolarization. Small suprathreshold current injections led to preferred spike generation at the resonance frequency. Bath application of ZD7288 or Cs(+) , inhibitors of the hyperpolarization-activated cation current (Ih ), transformed the resonance into low-pass behaviour, whereas the T-channel blockers mibefradil and Ni(2+) decreased the strength of the resonance. It is concluded that nRt cells have an Ih -mediated intrinsic frequency preference in the subthreshold voltage range that favours action potential generation in the delta-frequency band.

The effects of HCN and KLT ion channels on adaptation and refractoriness in a stochastic auditory nerve model.

An accurate model of auditory nerve fibers (ANFs) may assist in developing improved cochlear implant (CI) stimulation strategies. Previous studies have shown that the original Hodgkin-Huxley (HH) model may be better at describing nodes of Ranvier in ANFs than models for other mammalian axon types. However, the HH model is still unable to explain a number of phenomena observed in auditory nerve responses to CI stimulation such as adaptation to high-rate stimulation and the time course of relative refractoriness. Recent physiological investigations of ANFs have shown the presence of a number of ion channel types not considered in the previous modeling studies, including low-threshold potassium (KLT) channels and hyperpolarization-activated cation (HCN) channels. In this paper, we investigate inclusion of these ion channel types in a stochastic HH model of a single node of Ranvier. Simulation results for pulse trains with rates of 200, 800, and 2000 pulse/s suggests that both the KLT channels and HCN channels can produce adaptation in the spike rate. However, the adaptation due to KLT is restricted to higher stimulation rates, whereas the adaptation due to HCN is observed across all stimulation rates. Additionally, using pulse pairs it was found that KLT increased both the absolute and the relative refractory periods. HCN on its own increased just the relative refractory period, but produced a synergistic increase in the absolute refractory period when combined with KLT. Together these results argue strongly for the need to consider HCN and KLT channels when studying CI stimulation of ANFs.

Response of human thalamic neurons to high-frequency stimulation.

Thalamic deep brain stimulation (DBS) is an effective treatment for tremor, but the mechanisms of action remain unclear. Previous studies of human thalamic neurons to noted transient rebound bursting activity followed by prolonged inhibition after cessation of high frequency extracellular stimulation, and the present study sought to identify the mechanisms underlying this response. Recordings from 13 thalamic neurons exhibiting low threshold spike (LTS) bursting to brief periods of extracellular stimulation were made during surgeries to implant DBS leads in 6 subjects with Parkinson's disease. The response immediately after cessation of stimulation included a short epoch of burst activity, followed by a prolonged period of silence before a return to LTS bursting. A computational model of a population of thalamocortical relay neurons and presynaptic axons terminating on the neurons was used to identify cellular mechanisms of the observed responses. The model included the actions of neuromodulators through inhibition of a non-pertussis toxin sensitive K+ current (IKL), activation of a pertussis toxin sensitive K+ current (IKG), and a shift in the activation curve of the hyperpolarization-activated cation current (Ih). The model replicated well the measured responses, and the prolonged inhibition was associated most strongly with changes in IKG while modulation of IKL or Ih had minimal effects on post-stimulus inhibition suggesting that neuromodulators released in response to high frequency stimulation are responsible for mediating the post-stimulation bursting and subsequent long duration silence of thalamic neurons. The modeling also indicated that the axons of the model neurons responded robustly to suprathreshold stimulation despite the inhibitory effects on the soma. The findings suggest that during DBS the axons of thalamocortical neurons are activated while the cell bodies are inhibited thus blocking the transmission of pathological signals through the network and replacing them with high frequency regular firing.

Cerebellar Inhibitory Input to the Inferior Olive Decreases Electrical Coupling and Blocks Subthreshold Oscillations

GABAergic projection neurons in the cerebellar nuclei (CN) innervate the inferior olive (IO) that in turn is the source of climbing fibers targeting Purkinje neurons in the cerebellar cortex. Anatomical evidence suggests that CN synapses modulate electrical coupling between IO neurons. Invivo studies indicate that they are also involved in controlling synchrony and rhythmicity of IO neurons. Here, we demonstrate using virally targeted channelrhodopsin in the cerebellar nucleo-olivary neurons that synaptic input can indeed modulate both the strength and symmetry of electrical coupling between IO neurons and alter network activity. Similar synaptic modifications of electrical coupling are likely to occur in other brain regions, where rapid modification of the spatiotemporal features of the coupled networks is needed to adequately respond to behavioral demands.

Cholinergic Neurons Excite Cortically Projecting Basal Forebrain GABAergic Neurons

The basal forebrain (BF) plays an important role in the control of cortical activation and attention. Understanding the modulation of BF neuronal activity is a prerequisite to treat disorders of cortical activation involving BF dysfunction, such as Alzheimer's disease. Here we reveal the interaction between cholinergic neurons and cortically projecting BF GABAergic neurons using immunohistochemistry and whole-cell recordings in vitro. In GAD67-GFP knock-in mice, BF cholinergic (choline acetyltransferase-positive) neurons were intermingled with GABAergic (GFP(+)) neurons. Immunohistochemistry for the vesicular acetylcholine transporter showed that cholinergic fibers apposed putative cortically projecting GABAergic neurons containing parvalbumin (PV). In coronal BF slices from GAD67-GFP knock-in or PV-tdTomato mice, pharmacological activation of cholinergic receptors with bath application of carbachol increased the firing rate of large (>20 μm diameter) BF GFP(+) and PV (tdTomato+) neurons, which exhibited the intrinsic membrane properties of cortically projecting neurons. The excitatory effect of carbachol was blocked by antagonists of M1 and M3 muscarinic receptors in two subpopulations of BF GABAergic neurons [large hyperpolarization-activated cation current (Ih) and small Ih, respectively]. Ion substitution experiments and reversal potential measurements suggested that the carbachol-induced inward current was mediated mainly by sodium-permeable cation channels. Carbachol also increased the frequency of spontaneous excitatory and inhibitory synaptic currents. Furthermore, optogenetic stimulation of cholinergic neurons/fibers caused a mecamylamine- and atropine-sensitive inward current in putative GABAergic neurons. Thus, cortically projecting, BF GABAergic/PV neurons are excited by neighboring BF and/or brainstem cholinergic neurons. Loss of cholinergic neurons in Alzheimer's disease may impair cortical activation, in part, through disfacilitation of BF cortically projecting GABAergic/PV neurons.

HCN2 channels account for mechanical (but not heat) hyperalgesia during long-standing inflammation

There is emerging evidence that hyperpolarization-activated cation (HCN) channels are involved in the development of pathological pain, including allodynia and hyperalgesia. Mice lacking the HCN isoform 2 display reduced heat but unchanged mechanical pain behavior, as recently shown in preclinical models of acute inflammatory pain. However, the impact of HCN2 to chronic pain conditions is less clear and has not been examined so far. In this report, we study the role of HCN2 in the complete Freund’s adjuvant inflammation model reflecting chronic pain conditions. We used sensory neuron–specific as well as inducible global HCN2 mutants analyzing pain behavior in persistent inflammation and complemented this by region-specific administration of an HCN channel blocker. Our results demonstrate that the absence of HCN2 in primary sensory neurons reduces tactile hypersensitivity in chronic inflammatory conditions but leaves heat hypersensitivity unaffected. This result is in remarkable contrast to the recently described role of HCN2 in acute inflammatory conditions. We show that chronic inflammation results in an increased expression of HCN2 and causes sensitization in peripheral and spinal terminals of the pain transduction pathway. The contribution of HCN2 to peripheral sensitization mechanisms was further supported by single-fiber recordings from isolated skin–nerve preparations and by conduction velocity measurements of saphenous nerve preparations. Global HCN2 mutants revealed that heat hypersensitivity—unaffected in peripheral HCN2 mutants—was diminished by the additional disruption of central HCN2 channels, suggesting that thermal hyperalgesia under chronic inflammatory conditions is mediated by HCN2 channels beyond primary sensory afferents.

Subthreshold Resonance Oscillation and Generation of Action Potential

Subthreshold resonance has been observed in many excitatory/inhibitory neurons in the brain. It is suggested that such resonance phenomenon plays an important role in behavioral or perceptual functions in animals. However its mechanism and role remains unknown. Subthreshold resonance oscillation observed in a neuron suggests that a neuron has the frequency selectivity for various external inputs given to the brain. By advances in physiological experimental techniques, various voltage-dependent channels are thought to be involved in the generation of these resonance oscillations. Among these channels, a persistent sodium channel and a hyperpolarization-activated cation channel are said to mediate the generation of the subthreshold resonance oscillation observed in entorhinal cortex neurons and hippocampal pyramidal neurons. In this proposal, we examine how the external inputs influence the generation of action potentials at the neuron model with a persistent sodium channel and a hyperpolarization-activated cation channel parameters by using computer simulations. Furthermore, the role of DC bias current input in the neuron model is also discussed.

TRIP(8B)Ing up and Down HCN Channel Gating and Trafficking

The proper neuronal functioning of ion channels depends on their correct targeting to distinct polarized neuronal compartments, where the channels often mediate highly specific functions. HCN1 channels, which underlie the hyperpolarization-activated cation current (Ih) in many types of neurons, are targeted to the distal apical dendrites of hippocampal CA1 pyramidal neurons, where they regulate the integration of synaptic inputs and control excitability. Results from our laboratory and others indicate that the cytoplasmic protein TRIP8b is the major auxiliary subunit of HCN1 channels in the brain, where it plays an important role in regulating HCN1 function, expression and localization. TRIP8b undergoes extensive alternative splicing at its N-terminus, with at least 10 splice variants detected in brain. All splice variants interact strongly with the C-terminus of all four HCN channel isoforms (HCN1-4) at two different interaction surfaces. Whereas all TRIP8b isoforms inhibit channel gating by antagonizing the normal action of cAMP to facilitate opening, the various isoforms have distinct effects on channel trafficking. We identified two splice isoforms with opposing actions on HCN1 surface expression and distinct subcellular locales that are critical for HCN1 dendritic targeting. Our more recent results have identified the structural and functional bases for many of the regulatory actions of TRIP8b.

Activation of rhodopsin gene transcription in cultured retinal precursors of chicken embryo: role of Ca(2+) signaling and hyperpolarization-activated cation channels.

This study reports that the spontaneous 50-fold activation of rhodopsin gene transcription, observed in cultured retinal precursors from 13-day chicken embryo, relies on a Ca(2+)-dependent mechanism. Activation of a transiently transfected rhodopsin promoter (luciferase reporter) in these cells was inhibited (60%) by cotransfection of a dominant-negative form of the cAMP-responsive element-binding protein. Both rhodopsin promoter activity and rhodopsin mRNA accumulation were blocked by Ca(2+)/calmodulin-dependent kinase II inhibitors, but not by protein kinase A inhibitors, suggesting a role of Ca(2+) rather than cAMP. This was confirmed by the inhibitory effect of general and T-type selective Ca(2+) channel blockers. Oscillations in Ca(2+) fluorescence (Fluo8) could be observed in 1/10 cells that activated the rhodopsin promoter (DsRed reporter). A robust and reversible inhibition of rhodopsin gene transcription by ZD7288 indicated a role of hyperpolarization-activated channels (HCN). Cellular localization and developmental expression of HCN1 were compatible with a role in the onset of rhodopsin gene transcription. Together, the data suggest that the spontaneous activation of rhodopsin gene transcription in cultured retinal precursors results from a signaling cascade that involves the pacemaker activity of HCN channels, the opening of voltage-gated Ca(2+)-channels, activation of Ca(2+)/calmodulin-dependent kinase II and phosphorylation of cAMP-responsive element-binding protein. Rhodopsin gene expression in cultured retinal precursors from chicken embryo relies on a Ca2+-dependent mechanism whereby hyperpolarization-activated cyclic nucleotide-gated channels (HCN) activate T-type voltage-dependent Ca2+ channels (VDCC) through membrane depolarization, causing calmodulin-dependent kinase II (CaMKII) to phosphorylate the cAMP-responsive element-binding protein (CREB) and leading to activation of rhodopsin gene transcription. Photoreceptor localization and development of HCN1 channels suggest similar role in vivo.