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Circadian rhythms have been extensively studied in Drosophila ; however, still little is known about how the electrical properties of clock neurons are specified. We have performed a behavioral genetic screen through the downregulation of candidate ion channels in the lateral ventral neurons (LNvs) and show that the hyperpolarization-activated cation current I h is important for the behaviors that the LNvs influence: temporal organization of locomotor activity, analyzed in males, and sleep, analyzed in females. Using whole-cell patch clamp electrophysiology we demonstrate that small LNvs (sLNvs) are bursting neurons, and that I h is necessary to achieve the high-frequency bursting firing pattern characteristic of both types of LNvs in females. Since firing in bursts has been associated to neuropeptide release, we hypothesized that I h would be important for LNvs communication. Indeed, herein we demonstrate that I h is fundamental for the recruitment of pigment dispersing factor (PDF) filled dense core vesicles (DCVs) to the terminals at the dorsal protocerebrum and for their timed release, and hence for the temporal coordination of circadian behaviors. SIGNIFICANCE STATEMENT Ion channels are transmembrane proteins with selective permeability to specific charged particles. The rich repertoire of parameters that may gate their opening state, such as voltage-sensitivity, modulation by second messengers and specific kinetics, make this protein family a determinant of neuronal identity. Ion channel structure is evolutionary conserved between vertebrates and invertebrates, making any discovery easily translatable. Through a screen to uncover ion channels with roles in circadian rhythms, we have identified the I h channel as an important player in a subset of clock neurons of the fruit fly. We show that lateral ventral neurons (LNvs) need I h to fire action potentials in a high-frequency bursting mode and that this is important for peptide transport and the control of behavior., (Copyright © 2021 the authors.)

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed as four different isoforms (HCN1-4) in the heart and in the central and peripheral nervous systems. In the voltage range of activation, HCN channels carry an inward current mediated by Na + and K + , termed I f in the heart and I h in neurons. Altered function of HCN channels, mainly HCN4, is associated with sinus node dysfunction and other arrhythmias such as atrial fibrillation, ventricular tachycardia, and atrioventricular block. In recent years, several data have also shown that dysfunctional HCN channels, in particular HCN1, but also HCN2 and HCN4, can play a pathogenic role in epilepsy; these include experimental data from animal models, and data collected over genetic mutations of the channels identified and characterized in epileptic patients. In the central nervous system, alteration of the I h current could predispose to the development of neurodegenerative diseases such as Parkinson's disease; since HCN channels are widely expressed in the peripheral nervous system, their dysfunctional behavior could also be associated with the pathogenesis of neuropathic pain. Given the fundamental role played by the HCN channels in the regulation of the discharge activity of cardiac and neuronal cells, the modulation of their function for therapeutic purposes is under study since it could be useful in various pathological conditions. Here we review the present knowledge of the HCN-related channelopathies in cardiac and neurological diseases, including clinical, genetic, therapeutic, and physiopathological aspects.

In mammals, acoustic information arises in the cochlea and is transmitted to the ventral cochlear nuclei (VCN). Three groups of VCN neurons extract different features from the firing of auditory nerve fibers and convey that information along separate pathways through the brainstem. Two of these pathways process temporal information: octopus cells detect coincident firing among auditory nerve fibers and transmit signals along monaural pathways, and bushy cells sharpen the encoding of fine structure and feed binaural pathways. The ability of these cells to signal with temporal precision depends on a low-voltage-activated K + conductance (g KL ) and a hyperpolarization-activated conductance (g h ). This 'tale of two conductances' traces gap detection and sound lateralization to their cellular and biophysical origins., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

An important contribution to neural circuit oscillatory dynamics is the ongoing activation and inactivation of hyperpolarization-activated currents ( I h ). Network synchrony dynamics play an important role in the initial processing of odor signals by the main olfactory bulb (MOB) and accessory olfactory bulb (AOB). In the mouse olfactory bulb, we show that I h is present in granule cells (GCs), the most prominent inhibitory neuron in the olfactory bulb, and that I h underlies subthreshold resonance in GCs. In accord with the properties of I h , the currents exhibited sensitivity to changes in extracellular K + concentration and ZD7288 (4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidin chloride), a blocker of I h -dependent subthreshold resonance in the delta range (1-4 Hz), while principal neurons, the mitral cells, do not exhibit I h is active at rest in GCs. The inclusion of cAMP in the intracellular solution shifted the activation of I h to less negative potentials in the MOB, but not in the AOB, suggesting that channels with different subunit composition mediate I h in these regions. Furthermore, we show that mature GCs exhibit I h -dependent subthreshold resonance in the theta frequency range (4-12 Hz). Another inhibitory subtype in the MOB, the periglomerular cells, exhibited I h -dependent subthreshold resonance in the delta range (1-4 Hz), while principal neurons, the mitral cells, do not exhibit I h -dependent subthreshold resonance. Importantly, I h size, as well as the strength and frequency of resonance in GCs, exhibited a postnatal developmental progression, suggesting that this development of I h in GCs may differentially contribute to their integration of sensory input and contribution to oscillatory circuit dynamics.

Experimental and computational studies demonstrate that different sets of intrinsic and synaptic conductances can give rise to equivalent activity patterns. This is because the balance of conductances, not their absolute values, defines a given activity feature. Activity-dependent feedback mechanisms maintain neuronal conductance correlations and their corresponding activity features. This study demonstrates that tonic nM concentrations of monoamines enable slow, activity-dependent processes that can maintain a correlation between the transient potassium current (I(A) and the hyperpolarization activated current (Ih) over the long-term (i.e., regulatory change persists for hours after removal of modulator). Tonic 5 nM DA acted through an RNA interference silencing complex (RISC)- and RNA polymerase II-dependent mechanism to maintain a long-term positive correlation between I(A) and Ih in the lateral pyloric neuron (LP) but not in the pyloric dilator neuron (PD). In contrast, tonic 5 nM 5HT maintained a RISC-dependent positive correlation between I(A) and Ih in PD but not LP over the long-term. Tonic 5 nM OCT maintained a long-term negative correlation between I(A) and Ih in PD but not LP; however, it was only revealed when RISC was inhibited. This study also demonstrated that monoaminergic tone can also preserve activity features over the long-term: the timing of LP activity, LP duty cycle and LP spike number per burst were maintained by tonic 5 nM DA. The data suggest that low-level monoaminergic tone acts through multiple slow processes to permit cell-specific, activity-dependent regulation of ionic conductances to maintain conductance correlations and their corresponding activity features over the long-term.

Compelling evidence suggests that pathological activity of the external globus pallidus (GPe), a nucleus in the basal ganglia, contributes to the motor symptoms of a variety of movement disorders such as Parkinson's disease. Recent studies have challenged the idea that the GPe comprises a single, homogenous population of neurons that serves as a simple relay in the indirect pathway. However, we still lack a full understanding of the diversity of the neurons that make up the GPe. Specifically, a more precise classification scheme is needed to better describe the fundamental biology and function of different GPe neuron classes. To this end, we generated a novel multicistronic BAC (bacterial artificial chromosome) transgenic mouse line under the regulatory elements of the Npas1 gene. Using a combinatorial transgenic and immunohistochemical approach, we discovered that parvalbumin-expressing neurons and Npas1-expressing neurons in the GPe represent two nonoverlapping cell classes, amounting to 55% and 27% of the total GPe neuron population, respectively. These two genetically identified cell classes projected primarily to the subthalamic nucleus and to the striatum, respectively. Additionally, parvalbumin-expressing neurons and Npas1-expressing neurons were distinct in their autonomous and driven firing characteristics, their expression of intrinsic ion conductances, and their responsiveness to chronic 6-hydroxydopamine lesion. In summary, our data argue that parvalbumin-expressing neurons and Npas1-expressing neurons are two distinct functional classes of GPe neurons. This work revises our understanding of the GPe, and provides the foundation for future studies of its function and dysfunction., Significance Statement: Until recently, the heterogeneity of the constituent neurons within the external globus pallidus (GPe) was not fully appreciated. We addressed this knowledge gap by discovering two principal GPe neuron classes, which were identified by their nonoverlapping expression of the markers parvalbumin and Npas1. Our study provides evidence that parvalbumin and Npas1 neurons have different topologies within the basal ganglia., (Copyright © 2015 the authors 0270-6474/15/3511830-18$15.00/0.)

The actin-binding protein filamin A (FLNa) regulates neuronal migration during development, yet its roles in the mature brain remain largely obscure. Here, we probed the effects of FLNa on the regulation of ion channels that influence neuronal properties. We focused on the HCN1 channels that conduct Ih, a hyperpolarization-activated current crucial for shaping intrinsic neuronal properties. Whereas regulation of HCN1 channels by FLNa has been observed in melanoma cell lines, its physiological relevance to neuronal function and the underlying cellular pathways that govern this regulation remain unknown. Using a combination of mutational, pharmacological, and imaging approaches, we find here that FLNa facilitates a selective and reversible dynamin-dependent internalization of HCN1 channels in HEK293 cells. This internalization is accompanied by a redistribution of HCN1 channels on the cell surface, by accumulation of the channels in endosomal compartments, and by reduced Ih density. In hippocampal neurons, expression of a truncated dominant-negative FLNa enhances the expression of native HCN1. Furthermore, acute abrogation of HCN1-FLNa interaction in neurons, with the use of decoy peptides that mimic the FLNa-binding domain of HCN1, abolishes the punctate distribution of HCN1 channels in neuronal cell bodies, augments endogenous Ih, and enhances the rebound-response ("voltage-sag") of the neuronal membrane to transient hyperpolarizing events. Together, these results support a major function of FLNa in modulating ion channel abundance and membrane trafficking in neurons, thereby shaping their biophysical properties and function.

The spiral ganglion conveys afferent auditory information predominantly through a single class of type I neurons that receive signals from inner hair cell sensory receptors. These auditory primary afferents, like in other systems (Puopolo and Belluzzi, 1998; Gascon and Moqrich, 2010; Leao et al., 2012) possess a marked diversity in their electrophysiological features (Taberner and Liberman, 2005). Consistent with these observations, when the auditory primary afferents were assessed in neuronal explants separated from their peripheral and central targets it was found that individual neurons were markedly heterogeneous in their endogenous electrophysiological features. One aspect of this heterogeneity, obvious throughout the ganglion, was their wide range of excitability as assessed by voltage threshold measurements (Liu and Davis, 2007). Thus, while neurons in the base differed significantly from apical and middle neurons in their voltage thresholds, each region showed distinctly wide ranges of values. To determine whether the resting membrane potentials (RMPs) of these neurons correlate with the threshold distribution and to identify the ion channel regulatory elements underlying heterogeneous neuronal excitability in the ganglion, patch-clamp recordings were made from postnatal day (P5-8) murine spiral ganglion neurons in vitro. We found that RMP mirrored the tonotopic threshold distribution, and contributed an additional level of heterogeneity in each cochlear location. Pharmacological experiments further indicated that threshold and RMP was coupled through the Kv1 current, which had a dual impact on both electrophysiological parameters. Whereas, hyperpolarization-activated cationic channels decoupled these two processes by primarily affecting RMP without altering threshold level. Thus, beyond mechanical and synaptic specializations, ion channel regulation of intrinsic membrane properties imbues spiral ganglion neurons with different excitability levels, a feature that contributes to primary auditory afferent diversity., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

The "amyloid-β (Aβ) hypothesis" posits that accumulating Aβ peptides (Aβs) produced by neurons cause Alzheimer's disease (AD). However, the Aβs contribution by the more numerous astrocytes remains undetermined. Previously we showed that fibrillar (f)Aβ25-35, an Aβ42 proxy, evokes a surplus endogenous Aβ42 production/accumulation in cortical adult human astrocytes. Here, by using immunocytochemistry, immunoblotting, enzymatic assays, and highly sensitive sandwich ELISA kits, we investigated the effects of fAβ25-35 and soluble (s)Aβ25-35 on Aβ42 and Aβ40 accumulation/secretion by human cortical astrocytes and HCN-1A neurons and, since the calcium-sensing receptor (CaSR) binds Aβs, their modulation by NPS 2143, a CaSR allosteric antagonist (calcilytic). The fAβ25-35-exposed astrocytes and surviving neurons produced, accumulated, and secreted increased amounts of Aβ42, while Aβ40 also accrued but its secretion was unchanged. Accordingly, secreted Aβ42/Aβ40 ratio values rose for astrocytes and neurons. While slightly enhancing Aβ40 secretion by fAβ25-35-treated astrocytes, NPS 2143 specifically suppressed the fAβ25-35-elicited surges of endogenous Aβ42 secretion by astrocytes and neurons. Therefore, NPS 2143 addition always kept Aβ42/Aβ40 values to baseline or lower levels. Mechanistically, NPS 2143 decreased total CaSR protein complement, transiently raised proteasomal chymotrypsin activity, and blocked excess NO production without affecting the ongoing increases in BACE1/β-secretase and γ-secretase activity in fAβ25-35-treated astrocytes. Compared to fAβ25-35, sAβ25-35 also stimulated Aβ42 secretion by astrocytes and neurons and NPS 2143 specifically and wholly suppressed this effect. Therefore, since NPS 2143 thwarts any Aβ/CaSR-induced surplus secretion of endogenous Aβ42 and hence further vicious cycles of Aβ self-induction/secretion/spreading, calcilytics might effectively prevent/stop the progression to full-blown AD., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The actin-binding protein filamin A (FLNa) regulates neuronal migration during development, yet its roles in the mature brain remain largely obscure. Here, we probed the effects of FLNa on the regulation of ion channels that influence neuronal properties. We focused on the HCN1 channels that conduct Ih, a hyperpolarization-activated current crucial for shaping intrinsic neuronal properties. Whereas regulation of HCN1 channels by FLNa has been observed in melanoma cell lines, its physiological relevance to neuronal function and the underlying cellular pathways that govern this regulation remain unknown. Using a combination of mutational, pharmacological, and imaging approaches, we find here that FLNa facilitates a selective and reversible dynamin-dependent internalization of HCN1 channels in HEK293 cells. This internalization is accompanied by a redistribution of HCN1 channels on the cell surface, by accumulation of the channels in endosomal compartments, and by reduced Ih density. In hippocampal neurons, expression of a truncated dominant-negative FLNa enhances the expression of native HCN1. Furthermore, acute abrogation of HCN1-FLNa interaction in neurons, with the use of decoy peptides that mimic the FLNa-binding domain of HCN1, abolishes the punctate distribution of HCN1 channels in neuronal cell bodies, augments endogenous Ih, and enhances the rebound-response ("voltage-sag") of the neuronal membrane to transient hyperpolarizing events. Together, these results support a major function of FLNa in modulating ion channel abundance and membrane trafficking in neurons, thereby shaping their biophysical properties and function.

Aim: Acetylcholine release is vital in the pacing of theta rhythms in the hippocampus. The subiculum is the output region of the hippocampus with different neuronal subtypes that generate theta oscillations during arousal and rapid eye movement sleep. The combination of intrinsic resonance in the hippocampal neurons and the periodic excitation of hippocampal excitatory and inhibitory neurons by cholinergic pathway drives theta oscillations. However, the acetylcholine mediated effect on intrinsic subthreshold resonance generating hyperpolarization‐activated cyclic nucleotide‐gated current, Ih of subicular neurons is unexplored. We studied the acetylcholine receptor‐independent effect of cholinergic agents on the intrinsic properties of subiculum principal neurons and the underlying mechanism. Methods: We bath perfused acetylcholine or nicotine on rat brain slices in the presence of synaptic blockers. The physiological effect was studied by cholinergic fibres stimulation and electrophysiological recordings under whole‐cell mode of subiculum neurons using septohippocampal sections. Results: Exogenously applied acetylcholine in the presence of atropine affected two groups of subicular neurons differently. Acetylcholine reduced the resonance frequency and Ih in bursting neurons, whereas these properties were unaffected in regular firing neurons. Subsequently, the endogenously released acetylcholine by stimulation showed a selective suppressive effect on Ih, sag, and resonance in burst firing among the two excitatory neurons. Nicotine suppressed the Ih amplitude in burst firing neurons, which was evident by decreased sag amplitude and resonance frequency and increased excitability. Conclusion: Our study suggests cell type‐specific acetylcholine receptor‐independent shift in resonance frequency by partially inhibiting HCN current during high cholinergic inputs. [ABSTRACT FROM AUTHOR]

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are voltage-gated ion channels that critically modulate neuronal activity. Four HCN subunits (HCN1–4) have been cloned, each having a unique expression profile and distinctive effects on neuronal excitability within the brain. Consistent with this, the expression and function of these subunits are altered in diverse ways in neurological disorders. Here, we review current knowledge on the structure and distribution of the individual HCN channel isoforms, their effects on neuronal activity under physiological conditions, and how their expression and function are altered in neurological disorders, particularly epilepsy, neuropathic pain, and affective disorders. We discuss the suitability of HCN channels as therapeutic targets and how drugs might be strategically designed to specifically act on particular isoforms. We conclude that medicines that target individual HCN isoforms and/or their auxiliary subunit, TRIP8b, may provide valuable means of treating distinct neurological conditions. [ABSTRACT FROM AUTHOR]

Febrile seizures, in addition to being the most common seizure type of the developing human, may contribute to the generation of subsequent limbic epilepsy. Our previous work has demonstrated that prolonged experimental febrile seizures in the immature rat model increased hippocampal excitability long term, enhancing susceptibility to future seizures. The mechanisms for these profound proepileptogenic changes did not require cell death and were associated with long-term slowed kinetics of the hyperpolarization-activated depolarizing current (I(H)). Here we show that these seizures modulate the expression of genes encoding this current, the hyperpolarization-activated, cyclic nucleotide-gated channels (HCNs): In CA1 neurons expressing multiple HCN isoforms, the seizures induced a coordinated reduction of HCN1 mRNA and enhancement of HCN2 expression, thus altering the neuronal HCN phenotype. The seizure-induced augmentation of HCN2 expression involved CA3 in addition to CA1, whereas for HCN4, mRNA expression was not changed by the seizures in either hippocampal region. This isoform- and region-specific transcriptional regulation of the HCNs required neuronal activity rather than hyperthermia alone, correlated with seizure duration, and favored the formation of slow-kinetics HCN2-encoded channels. In summary, these data demonstrate a novel, activity-dependent transcriptional regulation of HCN molecules by developmental seizures. These changes result in long-lasting alteration of the HCN phenotype of specific hippocampal neuronal populations, with profound consequences on the excitability of the hippocampal network.

Although conduction failure has been observed in nociceptive C-fibers, little is known regarding its significance or therapeutic potential. In a previous study, we demonstrated that C-fiber conduction failure, which is regarded as an intrinsic self-inhibition mechanism, was reduced in circumstances of painful diabetic neuropathy. In this study, we extend this finding in the complete Freund's adjuvant model of inflammatory pain and validate that the degree of conduction failure decreased and led to a greater amount of pain signals conveyed to the central nervous system. In complete Freund's adjuvant-injected animals, conduction failure occurred in a C-fiber-selective, activity-dependent manner and was associated with an increase in the rising slope of the C-fiber after-hyperpolarization potential. To target conduction failure in a therapeutic modality, we used ZD7288, an antagonist of hyperpolarization-activated, cyclic nucleotide-modulated channels which are activated by hyperpolarization and play a pivotal role in both inflammatory and neuropathic pain. ZD7288 promoted conduction failure by suppressing Ih as a mechanism to reduce the rising slope of the after-hyperpolarization potential. Moreover, perineuronal injection of ZD7288 inhibited abnormal mechanical allodynia and thermal hyperalgesia without affecting motor function or heart rate. Our data highlight the analgesic potential of local ZD7288 application and identify conduction failure as a novel target for analgesic therapeutic development. [ABSTRACT FROM AUTHOR]

Neuropeptide S (NPS), an endogenous anxiolytic, has been shown to protect against chronic pain through interacting with its cognate NPS receptor (NPSR) in the brain. However, the cellular mechanism of this NPS action remains unclear. We report that NPS inhibits hyperpolarization-activated cyclic nucleotide-gated (HCN) channel current ( Ih ) in the rat's amygdala through activation of NPSR. This NPS effect is mediated through ERK1/2 phosphorylation in a subset of pyramidal-like neurons located in the medial amygdala. The characters of the recorded I h suggest a major role for HCN1 activity in this process. Inhibition of I h by NPS stimulates the glutamatergic drive onto fast spiking intra-amygdalolidal GABAergic interneurons, which in turn facilitates GABA release onto pyramidal-like neurons. Moreover, the HCN1 expression is increased in the amygdala of rats with peripheral nerve injury and intra-amygdaloidal administration of the HCN channel inhibitor ZD7288 attenuates nociceptive behavior in these rats. These results suggest that NPS-mediated modulation of intra-amygdaloidal HCN channel activities may be an important central inhibitory mechanism for regulation of chronic pain. [ABSTRACT FROM AUTHOR]

Genetic ablation of cellular prion protein (PrPC) has been linked to increased neuronal excitability and synaptic activity in the hippocampus. We have previously shown that synaptic activity in hippocampi of PrP-null mice is increased due to enhanced N-methyl-D-aspartate receptor (NMDAR) function. Here, we focused on the effect of PRNP gene knock-out (KO) on intrinsic neuronal excitability, and in particular, the underlying ionic mechanism in hippocampal neurons cultured from P0 mouse pups. We found that the absence of PrPC profoundly affected the firing properties of cultured hippocampal neurons in the presence of synaptic blockers. The membrane impedance was greater in PrP-null neurons, and this difference was abolished by the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker ZD7288 (100 μM). HCN channel activity appeared to be functionally regulated by PrPC. The amplitude of voltage sag, a characteristic of activating HCN channel current (Ih), was decreased in null mice. Moreover, Ih peak current was reduced, along with a hyperpolarizing shift in activation gating and slower kinetics. However, neither HCN1 nor HCN2 formed a biochemical complex with PrPC. These results suggest that the absence of PrP downregulates the activity of HCN channels through activation of a cell signaling pathway rather than through direct interactions. This in turn contributes to an increase in membrane impedance to potentiate neuronal excitability. [ABSTRACT FROM AUTHOR]

A hallmark of peripheral neuropathic pain (PNP) is chronic spontaneous pain and/or hypersensitivity to normally painful stimuli (hyperalgesia) or normally nonpainful stimuli (allodynia).This pain results partly from abnormal hyperexcitability of dorsal root ganglion (DRG) neurons. We have previously shown, using a modified version of the lumbar 5 (L5)-spinal nerve ligation model of PNP (mSNA model involving L5-spinal nerve axotomy plus loose ligation of the lumbar 4 (L4)-spinal nerve with neuroinflammation-inducing chromic-gut), that L4 DRG neurons exhibit increased spontaneous activity, the key characteristic of neuronal hyperexcitability. The underlying ionic and molecular mechanisms of the hyperexcitability of L4 DRG neurons are incompletely understood, but could result from changes in expression and/or function of ion channels including hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which are active near the neuron’s resting membrane potential, and which produce an excitatory inward current that depolarizes the membrane potential toward the threshold of action potential generation. Therefore, in the present study we used the mSNA model to investigate whether: (a) expression of HCN1–HCN3 channels is altered in L4 DRG neurons which, in the mSNA model, are essential for transmission of the evoked pain, and which contribute to chronic spontaneous pain, and (b) local (intraplantar) blockade of these HCN channels, with a specific blocker, ZD7288, attenuates chronic spontaneous pain and/or evoked pain in mSNA rats. We found 7 days after mSNA: (1) a significant increase in HCN2-immunoreactivity in small (<30 μm) DRG neurons (predominantly IB4-negative neurons), and in the proportion of small neurons expressing HCN2 (putative nociceptors); (2) no significant change in HCN1- or HCN3-immunoreactivity in all cell types; and (3) attenuation, with ZD7288 (100 μM intraplantar), of chronic spontaneous pain behavior (spontaneous foot lifting) and mechanical, but not, heat hypersensitivity. The results suggest that peripheral HCN channels contribute to mechanisms of spinal nerve injury-induced PNP, and that HCN channels, possibly HCN2, represent a novel target for PNP treatment. [ABSTRACT FROM AUTHOR]

Burst-firing in distinct subsets of thalamic relay (TR) neurons is thought to be a key requirement for the propagation of absence seizures. However, in the well-regarded Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model as yet there has been no link described between burst-firing in TR neurons and spike-and-wave discharges (SWDs). GAERS ventrobasal (VB) neurons are a specific subset of TR neurons that do not normally display burst-firing during absence seizures in the GAERS model, and here, we assessed the underlying relationship of VB burst-firing with I and T-type calcium currents between GAERS and non-epileptic control (NEC) animals. In response to 200-ms hyperpolarizing current injections, adult epileptic but not pre-epileptic GAERS VB neurons displayed suppressed burst-firing compared to NEC. In response to longer duration 1,000-ms hyperpolarizing current injections, both pre-epileptic and epileptic GAERS VB neurons required significantly more hyperpolarizing current injection to burst-fire than those of NEC animals. The current density of the Hyperpolarization and Cyclic Nucleotide-activated (HCN) current (I) was found to be increased in GAERS VB neurons, and the blockade of I relieved the suppressed burst-firing in both pre-epileptic P15-P20 and adult animals. In support, levels of HCN-1 and HCN-3 isoform channel proteins were increased in GAERS VB thalamic tissue. T-type calcium channel whole-cell currents were found to be decreased in P7-P9 GAERS VB neurons, and also noted was a decrease in Ca3.1 mRNA and protein levels in adults. Z944, a potent T-type calcium channel blocker with anti-epileptic properties, completely abolished hyperpolarization-induced VB burst-firing in both NEC and GAERS VB neurons. [ABSTRACT FROM AUTHOR]

Bursting behavior of brainstem premotor burst neurons (BNs) is essential for initiation of saccades and calibrating their metrics. Several ion channel families such as voltage-gated potassium (Kv) channels, low-voltage-activated calcium (Cav3) channels and hyperpolarization-activated cyclic nucleotide–gated (HCN) channels are major regulators of the bursting in neurons. Therefore, it was speculated that ion channels with rapid kinematics are essential for characteristic firing patterns of the BNs and rapid saccade velocities. However, the expression patterns of ion channels are yet to be confirmed. Confirmation would not only support the neuromimetic model predictions for saccade generation in brainstem, but also contemporary views that channelopathies can cause saccade disorders in humans. As proof of concept, we examined excitatory BNs in the rostral interstitial nucleus of medial longitudinal fasciculus (RIMLF, vertical saccades) and inhibitory BNs in nucleus paragigantocellularis dorsalis (PGD, horizontal saccades) histochemically in macaque monkeys. We found strong expression of Kv channels, which enable rapid-firing, as well as HCN1&2 and Cav3.2&3.3, which enable post-inhibitory rebound bursting, in both BN populations. Moreover, PGD was found to host multiple neuron groups in terms of calretinin immunoreactivity. Our results provide histochemical evidence that supports models proposing post-inhibitory rebound facilitates bursting in BNs. Furthermore, our findings support the notion that deductions can be made about electrophysiological firing properties by histochemical examination of functional groups within the brainstem saccadic circuitry. This development is an important building block supporting the concept of channelopathies in saccadic disorders. Future histological studies in humans will confirm this approach for saccadic disorders. • Ion channel immunostaining of saccadic burst neurons was studied in macaque monkey. • Excitatory and inhibitory burst neurons express Kv1 & Kv3 channels for fast firing. • Burst neurons express HCN1&2 and Cav3.2&3.3 facilitating post-inhibitory rebound. • Important step in the inquiry of channelopathies as a cause of saccadic disorders. [ABSTRACT FROM AUTHOR]

Experiments in rodent models of Parkinson's disease have demonstrated a prominent increase of oscillatory firing patterns in neurons within the Parkinsonian globus pallidus (GP) which may underlie some of the motor symptoms of the disease. There are two main pathways from the cortex to GP: via the striatum and via the subthalamic nucleus (STN), but it is not known how these inputs sculpt the pathological pallidal firing patterns. To study this we developed a novel neural network model of conductance-based spiking pallidal neurons with cortex-modulated input from STN neurons. Our results support the hypothesis that entrainment occurs primarily via the subthalamic pathway. We find that as a result of the interplay between excitatory input from the STN and mutual inhibitory coupling between GP neurons, a homogeneous population of GP neurons demonstrates a self-organizing dynamical behavior where two groups of neurons emerge: one spiking in-phase with the cortical rhythm and the other in anti-phase. This finding mirrors what is seen in recordings from the GP of rodents that have had Parkinsonism induced via brain lesions. Our model also includes downregulation of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels in response to burst firing of GP neurons, since this has been suggested as a possible mechanism for the emergence of Parkinsonian activity. We found that the downregulation of HCN channels provides even better correspondence with experimental data but that it is not essential in order for the two groups of oscillatory neurons to appear.We discuss how the influence of inhibitory striatal input will strengthen our results. [ABSTRACT FROM AUTHOR]

Background: During neocortical development, multiple voltage- and ligand-gated ion channels are differentially expressed in neurons thereby shaping their intrinsic electrical properties. One of these voltage-gated ion channels, the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel and its current /h, is an important regulator of neuronal excitability. Thus far, studies on an early /h appearance in rodent neocortex are missing or conflicting. Therefore, we focused our study on perinatal neocortical /h and its properties. Results: In the perinatal rat neocortex we observed a rapid increase in the number of neurons exhibiting /h. Perinatal /h had unique properties: first, a pronounced cAMP sensitivity resulting in a marked shift of the voltage sufficient for half-maximum activation of the current towards depolarized voltages and second, an up to 10 times slower deactivation at physiological membrane potentials when compared to the one at postnatal day 30. The combination of these features was sufficient to suppress membrane resonance in our in silico and in vitro experiments. Although all four HCN subunits were present on the mRNA level we only detected HCN4, HCN3 and HCN1 on the protein level at P0. HCN1 protein at P0, however, appeared incompletely processed. At P30 glycosilated HCN1 and HCN2 dominated. By in silico simulations and heterologous co-expression experiments of a 'slow' and a 'fast' /h conducting HCN channel subunit in HEK293 cells, we mimicked most characteristics of the native current, pointing to a functional combination of subunit homo- or heteromeres. Conclusion: Taken together, these data indicate a HCN subunit shift initiated in the first 24 hours after birth and implicate a prominent perinatal role of the phylogenetically older HCN3 and/or HCN4 subunits in the developing neocortex. [ABSTRACT FROM AUTHOR]

Hyperpolarization-activated cyclic nucleotide-gated ion channels (h-channels; Ih ; HCN) modulate intrinsic excitability in hippocampal and neocortical pyramidal neurons, among others. Whereas Ih mediated by the HCN2 isoform is regulated by cAMP, there is little known about kinase modulation of Ih, especially for the HCN1 isoform predominant in pyramidal neurons. We used a computational method to identify a novel kinase modulator of h-channels, p38 mitogen-activated protein kinase (p38 MAPK). Inhibition of p38 MAPK in hippocampal pyramidal neurons caused a ∼25 mV hyperpolarization of Ih voltage-dependent activation. This downregulation of Ih produced hyperpolarization of resting potential, along with increased input resistance and temporal summation of excitatory inputs. Activation of p38 MAPK caused a ∼11 mV depolarizing shift in Ih activation, along with depolarized resting potential, and decreased input resistance and temporal summation. Inhibition of related MAPKs, ERK 1/2 (extracellular signal-related kinase 1/2) and JNK (c-Jun N-terminal kinase), produced no effect on Ih. These results show that p38 MAPK is a strong modulator of h-channel biophysical properties and may deserve additional exploration as a link between altered Ih and pathological conditions such as epilepsy. [ABSTRACT FROM AUTHOR]

Purpose: The hyperpolarization-activated cation current (IH) has been proposed to play a role in some forms of epileptogenesis, as it critically regulates synaptic integration and intrinsic excitability of principal limbic neurons and can be pathologically altered after experimentally induced seizures. In hippocampal CA1 pyramidal neurons, IH is functionally decreased after kainate-induced status epilepticus in adult rats but is increased after hyperthermia-induced seizures in immature rat pups. This study aimed to determine whether and how IH may be altered in CA1 pyramidal neurons after seizure-inducing global hypoxia in the neonatal brain. Methods: Seizures were induced in rat pups on postnatal day 10 by 14- to 16-min exposure to 5–7% O2. Whole-cell patch-clamp recordings were obtained from hippocampal CA1 pyramidal neurons in slices 30 min to 3 days after hypoxia treatment, and from control age-matched littermates. IH was isolated under voltage-clamp by subtracting current responses to hyperpolarizing voltage steps before and during application of the IH blocker ZD 7288 (100 μ M). Results: IH was significantly decreased in pyramidal neurons from the hypoxia-treated group compared with controls (p < 0.001; 19 controls; 15 hypoxia). Analyses of tail currents and activation kinetics indicated no statistically significant differences between groups in the voltage dependence or time constants of activation. Conclusions: These data indicate that a single episode of neonatal hypoxia that induces seizures can persistently decrease IH in CA1 pyramidal neurons, raising this as a potential contributing mechanism to epileptogenesis in this setting. Our findings further indicate that the consequences of seizures for IH may depend more on seizure etiology than on maturational stage. [ABSTRACT FROM AUTHOR]

The hyperpolarization-activated cation current (Ih) plays an important role in regulating neuronal excitability, yet its native single-channel properties in the brain are essentially unknown. Here we use variance-mean analysis to study the properties of single Ih channels in the apical dendrites of cortical layer 5 pyramidal neurons in vitro. In these neurons, we find that Ih channels have an average unitary conductance of 680 ± 30 fS (n=18). Spectral analysis of simulated and native Ih channels showed that there is little or no channel flicker below 5 kHz. In contrast to the uniformly distributed single-channel conductance, Ih channel number increases exponentially with distance, reaching densities as high as ∼550 channels/µm² at distal dendritic sites. These high channel densities generate significant membrane voltage noise. By incorporating a stochastic model of Ih single-channel gating into a morphologically realistic model of a layer 5 neuron, we show that this channel noise is higher in distal dendritic compartments and increased threefold with a 10-fold increased single-channel conductance (6.8 pS) but constant Ih current density. In addition, we demonstrate that voltage fluctuations attributable to stochastic Ih channel gating impact on action potential output, with greater spike-timing precision in models with the experimentally determined single-channel conductance. These data suggest that, in the face of high current densities, the small single-channel conductance of Ih is critical for maintaining the fidelity of action potential output. [ABSTRACT FROM AUTHOR]

Although the depressant effects of the general anesthetic propofol on thalamocortical relay neurons clearly involve γ-aminobutyric acid (GABA)A receptors, other mechanisms may be involved. The hyperpolarization-activated cation current ( Ih) regulates excitability and rhythmic firing in thalamocortical relay neurons in the ventrobasal (VB) complex of the thalamus. Here we investigated the effects of propofol on Ih-related function in vitro and in vivo. In whole-cell current-clamp recordings from VB neurons in mouse (P23–35) brain slices, propofol markedly reduced the voltage sag and low-threshold rebound excitation that are characteristic of the activation of Ih. In whole-cell voltage-clamp recordings, propofol suppressed the Ih conductance and slowed the kinetics of activation. The block of Ih by propofol was associated with decreased regularity and frequency of δ-oscillations in VB neurons. The principal source of the Ih current in these neurons is the hyperpolarization-activated cyclic nucleotide-gated (HCN) type 2 channel. In human embryonic kidney (HEK)293 cells expressing recombinant mouse HCN2 channels, propofol decreased Ih and slowed the rate of channel activation. We also investigated whether propofol might have persistent effects on thalamic excitability in the mouse. Three hours following an injection of propofol sufficient to produce loss-of-righting reflex in mice (P35), Ih was decreased, and this was accompanied by a corresponding decrease in HCN2 and HCN4 immunoreactivity in thalamocortical neurons in vivo. These results suggest that suppression of Ih may contribute to the inhibition of thalamocortical activity during propofol anesthesia. Longer-term effects represent a novel form of propofol-mediated regulation of Ih. [ABSTRACT FROM AUTHOR]

The globus pallidus (GP) is a critical component of the basal ganglia circuitry controlling motor behavior. Dysregulation of GP activity has been implicated in a number of psychomotor disorders, including Parkinson's disease (PD), in which a cardinal feature of the pathophysiology is an alteration in the pattern and synchrony of discharge in GP neurons. Yet the determinants of this activity in GP neurons are poorly understood. To help fill this gap, electrophysiological, molecular, and computational approaches were used to identify and characterize GABAergic GP neurons in tissue slices from rodents. In vitro, GABAergic GP neurons generate a regular, autonomous, single-spike pacemaker activity. Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels make an important contribution to this process: their blockade with ZD7288 significantly slowed discharge rate and decreased its regularity. HCN currents evoked by somatic voltage clamp had fast and slow components. Single-cell RT-PCR and immunohistochemical approaches revealed robust expression of HCN2 subunits as well as significant levels of HCN1 subunits in GABAergic GP neurons. Transient activation of striatal GABAergic input to GP neurons led to a resetting of rhythmic discharge that was dependent on HCN currents. Simulations suggested that the ability of transient striatal GABAergic input to reset pacemaking was dependent on dendritic HCN2/HCN1 channels. Together, these studies show that HCN channels in GABAergic GP neurons are key determinants of the regularity and rate ofpacemaking as well as striatal resetting of this activity, implicating HCN channels in the emergence of synchrony in PD. [ABSTRACT FROM AUTHOR]