Channel-based reporters for cAMP detection.

In the last 15 years, tremendous progress has been made in the development of single-cell cAMP sensors. Sensors are based upon cAMP-binding proteins that have been modified to transduce cAMP concentrations into electrical or fluorescent readouts that can be readily detected using patch clamp amplifiers, photomultiplier tubes, or cameras. Here we describe two complementary approaches for the detection and measurement of cAMP signals near the plasma membrane of cells. These probes take advantage of the ability of cyclic nucleotide-gated (CNG) channels to transduce small changes in cAMP concentrations into ionic flux through channel pores that can be readily detected by measuring Ca(2+) and/or Mn(2+) influx or by measuring ionic currents.

HCN channels: function and clinical implications.

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels belong to the superfamily of pore-loop cation channels. In mammals, the HCN channel family comprises 4 members (HCN1-4) that are expressed in heart and nervous system. HCN channels are activated by membrane hyperpolarization, are permeable to Na+ and K+, and are constitutively open at voltages near the resting membrane potential. In many cases, activation is facilitated by direct interaction with cyclic nucleotides, particularly cyclic adenosine monophosphate (cAMP). The cation current through HCN channels is known as I(h); opening of HCN channels elicits membrane depolarization toward threshold for action potential generation, and reduces membrane resistance and thus the magnitude of excitatory and inhibitory postsynaptic potentials. HCN channels have a major role in controlling neuronal excitability, dendritic integration of synaptic potentials, synaptic transmission, and rhythmic oscillatory activity in individual neurons and neuronal networks. These channels participate in mechanisms of synaptic plasticity and memory, thalamocortical rhythms, and somatic sensation. Experimental evidence indicates that HCN channels may also contribute to mechanisms of epilepsy and pain. The physiologic functions of HCN channels and their implications for neurologic disorders have been recently reviewed.(1-10).

Chronic inflammatory pain is associated with increased excitability and hyperpolarization-activated current (Ih) in C- but not Aδ-nociceptors.

Inflammatory pain hypersensitivity results partly from hyperexcitability of nociceptive (damage-sensing) dorsal root ganglion (DRG) neurons innervating inflamed tissue. However, most of the evidence for this is derived from experiments using acute inflammatory states. Herein, we used several approaches to examine the impact of chronic or persistent inflammation on the excitability of nociceptive DRG neurons and on their expression of I(h) and the underlying hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which regulate neuronal excitability. Using in vivo intracellular recordings of somatic action potentials from L4/L5 DRG neurons in normal rats and rats with hindlimb inflammation induced by complete Freund's adjuvant (CFA), we demonstrate increased excitability of C- but not Aδ-nociceptors, 5 to 7 days after CFA. This included an afterdischarge response to noxious pinch, which may contribute to inflammatory mechanohyperalgesia, and increased incidence of spontaneous activity (SA) and decreased electrical thresholds, which are likely to contribute to spontaneous pain and nociceptor sensitization, respectively. We also show, using voltage clamp in vivo, immunohistochemistry and behavioral assays that (1) the inflammation-induced nociceptor hyperexcitability is associated, in C- but not Aδ-nociceptors, with increases in the mean I(h) amplitude/density and in the proportion of I(h) expressing neurons, (2) increased proportion of small DRG neurons (mainly IB4-negative) expressing HCN2 but not HCN1 or HCN3 channel protein, (3) increased HCN2- immunoreactivity in the spinal dorsal horn, and (4) attenuation of inflammatory mechanoallodynia with the selective I(h) antagonist, ZD7288. Taken together, the findings suggest that C- but not Aδ-nociceptors sustain chronic inflammatory pain and that I(h)/HCN2 channels contribute to inflammation-induced C-nociceptor hyperexcitability.

Blocking effects of acehytisine on pacemaker currents (I(f)) in sinoatrial node cells and human HCN4 channels expressed in Xenopus laevis oocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Aconitum coreanum (Levl.) Raipaics has been extensively used to treat various kinds of disorders including cardiovascular disease in China for a long time. According to recent studies, its antiarrhythmic actions are attributable to the active component, acehytisine. However, the underlying mechanism remains poorly understood. AIM OF THE STUDY: The effects of acehytisine on the spontaneous activity in sinoatrial nodes and the electropharmacological action of this drug on I(f) in pacemaker cells and hHCN4 channels in oocytes were to be investigated. MATERIALS AND METHODS: Sinoatrial nodes were cut from rabbit heart, and transmembrane potentials were recorded by standard microelectrode technique. A whole-cell patch clamp technique was employed to record I(f) isolated enzymatically from rabbit sinoatrial node pacemaker cells. Human HCN4 channels were heterologously expressed in Xenopus oocytes and studied using the two-electrode voltage clamp technique. RESULTS: Acehytisine decreased the pacemaker rate of firing and slope of diastolic depolarization, modified the action potential configurations and blocked I(f) in rabbit sinoatrial node cells and hHCN4 channels expressed in Xenopus oocytes in a concentration-dependent, voltage-independent and non-use-dependent manner. Its electropharmacological properties were consistent with those of a close-state blocker. CONCLUSION: Our findings are likely to shed light on the clinical application of acehytisine in the treatment of cardiovascular disorders.

Corticospinal-specific HCN expression in mouse motor cortex: I(h)-dependent synaptic integration as a candidate microcircuit mechanism involved in motor control.

Motor cortex is a key brain center involved in motor control in rodents and other mammals, but specific intracortical mechanisms at the microcircuit level are largely unknown. Neuronal expression of hyperpolarization-activated current (I(h)) is cell class specific throughout the nervous system, but in neocortex, where pyramidal neurons are classified in various ways, a systematic pattern of expression has not been identified. We tested whether I(h) is differentially expressed among projection classes of pyramidal neurons in mouse motor cortex. I(h) expression was high in corticospinal neurons and low in corticostriatal and corticocortical neurons, a pattern mirrored by mRNA levels for HCN1 and Trip8b subunits. Optical mapping experiments showed that I(h) attenuated glutamatergic responses evoked across the apical and basal dendritic arbors of corticospinal but not corticostriatal neurons. Due to I(h), corticospinal neurons resonated, with a broad peak at ∼4 Hz, and were selectively modulated by α-adrenergic stimulation. I(h) reduced the summation of short trains of artificial excitatory postsynaptic potentials (EPSPs) injected at the soma, and similar effects were observed for short trains of actual EPSPs evoked from layer 2/3 neurons. I(h) narrowed the coincidence detection window for EPSPs arriving from separate layer 2/3 inputs, indicating that the dampening effect of I(h) extended to spatially disperse inputs. To test the role of corticospinal I(h) in transforming EPSPs into action potentials, we transfected layer 2/3 pyramidal neurons with channelrhodopsin-2 and used rapid photostimulation across multiple sites to synaptically drive spiking activity in postsynaptic neurons. Blocking I(h) increased layer 2/3-driven spiking in corticospinal but not corticostriatal neurons. Our results imply that I(h)-dependent synaptic integration in corticospinal neurons constitutes an intracortical control mechanism, regulating the efficacy with which local activity in motor cortex is transferred to downstream circuits in the spinal cord. We speculate that modulation of I(h) in corticospinal neurons could provide a microcircuit-level mechanism involved in translating action planning into action execution.

High frequency stimulation abolishes thalamic network oscillations: an electrophysiological and computational analysis.

Deep brain stimulation (DBS) of the thalamus has been demonstrated to be effective for the treatment of epilepsy. To investigate the mechanism of action of thalamic DBS, we examined the effects of high frequency stimulation (HFS) on spindle oscillations in thalamic brain slices from ferrets. We recorded intracellular and extracellular electrophysiological activity in the nucleus reticularis thalami (nRt) and in thalamocortical relay (TC) neurons in the lateral geniculate nucleus, stimulated the slice using a concentric bipolar electrode, and recorded the level of glutamate within the slice. HFS (100 Hz) of TC neurons generated excitatory post-synaptic potentials, increased the number of action potentials in both TC and nRt neurons, reduced the input resistance, increased the extracellular glutamate concentration, and abolished spindle wave oscillations. HFS of the nRt also suppressed spindle oscillations. In both locations, HFS was associated with significant and persistent elevation in extracellular glutamate levels and suppressed spindle oscillations for many seconds after the cessation of stimulation. We simulated HFS within a computational model of the thalamic network, and HFS also disrupted spindle wave activity, but the suppression of spindle activity was short-lived. Simulated HFS disrupted spindle activity for prolonged periods of time only after glutamate release and glutamate-mediated activation of a hyperpolarization-activated current (I(h)) was incorporated into the model. Our results suggest that the mechanism of action of thalamic DBS as used in epilepsy may involve the prolonged release of glutamate, which in turn modulates specific ion channels such as I(h), decreases neuronal input resistance, and abolishes thalamic network oscillatory activity.

Altered synaptic plasticity and behavioral abnormalities in CNGA3-deficient mice.

The role of the cyclic nucleotide-gated (CNG) channel CNGA3 is well established in cone photoreceptors and guanylyl cyclase-D-expressing olfactory neurons. To assess a potential function of CNGA3 in the mouse amygdala and hippocampus, we examined synaptic plasticity and performed a comparative analysis of spatial learning, fear conditioning and step-down avoidance in wild-type mice and CNGA3 null mutants (CNGA3(-/-) ). CNGA3(-/-) mice showed normal basal synaptic transmission in the amygdala and the hippocampus. However, cornu Ammonis (CA1) hippocampal long-term potentiation (LTP) induced by a strong tetanus was significantly enhanced in CNGA3(-/-) mice as compared with their wild-type littermates. Unlike in the hippocampus, LTP was not significantly altered in the amygdala of CNGA3(-/-) mice. Enhanced hippocampal LTP did not coincide with changes in hippocampus-dependent learning, as both wild-type and mutant mice showed a similar performance in water maze tasks and contextual fear conditioning, except for a trend toward higher step-down latencies in a passive avoidance task. In contrast, CNGA3(-/-) mice showed markedly reduced freezing to the conditioned tone in the amygdala-dependent cued fear conditioning task. In conclusion, our study adds a new entry on the list of physiological functions of the CNGA3 channel. Despite the dissociation between physiological and behavioral parameters, our data describe a so far unrecognized role of CNGA3 in modulation of hippocampal plasticity and amygdala-dependent fear memory.

Hyperpolarization-activated cation currents in human epileptogenic neocortex.

PURPOSE: Hyperpolarization-activated cation currents (I(H)) play a pivotal role in the control of neuronal excitability. In animal models of epilepsy both increases and decreases of I(H) have been reported. We, therefore, characterized properties of I(H) in human epileptogenic neocortex. METHODS: Layer II/III neurons in slices from epilepsy surgery tissues and rat cortex were investigated with whole-cell patch-clamp recordings. RESULTS: A total of 484 neurons from 96 temporal lobe epilepsy (TLE) tissues and 32 neurons from 8 frontal lobe epilepsy (FLE) tissues were recorded. Voltage-clamp recordings revealed on hyperpolarizing command steps two time- and voltage-dependent inward currents, namely a fast, Ba(2+)-sensitive current (K(IR)) and a slowly activating current, namely consisting of two kinetically distinct components sensitive to the established I(H) blocker ZD7288. Only, the fast component (I(H)(fast)) of TLE neurons was on average smaller and activated more slowly (density 2.7 +/- 1.6 pA/pF; tau 38.4 +/- 34.0 ms) than in FLE neurons (4.7 +/- 2.3 pA/pF; 16.6 +/- 7.9 ms; p < 0.001 for both). Within the TLE tissues the I(H)(fast) density (averaged per patient) was smaller in cases with numerous annual grand mal seizures (GM; 2.2 +/- 0.6 pA/pF) compared to those with few GM (2.8 +/- 1.0 pA/pF; p = 0.0184). A similar difference was obtained in the case of complex partial seizures (CPS; many CPS 2.2 +/- 0.6 pA/pF; few CPS 2.9 +/- 1.0 pA/pF, p = 0.0037). DISCUSSION: The biophysical properties of I(H) in cortices from TLE, FLE, and rat tissue suggest a deficit of HCN1 subunits in the human epileptogenic neocortex, which in turn may increase excitability and probability of seizure activity.

Sensitivity of HCN channel deactivation to cAMP is amplified by an S4 mutation combined with activation mode shift.

Hyperpolarisation-activation of HCN ion channels relies on the movement of a charged S4 transmembrane helix, preferentially stabilising the open conformation of the ion pore gate. The open state is additionally stabilised, (a) when cyclic AMP (cAMP) is bound to a cytoplasmic C-terminal domain or (b) when the "mode I" open state formed initially by gate opening undergoes a "mode shift" into a "mode II" open state with a new S4 conformation. We isolated a mutation (lysine 381 to glutamate) in S4 of mouse HCN4; patch-clamp of homomeric channels in excised inside-out membranes revealed a conditional phenotype. When cAMP-liganded K381E channels are previously activated by hyperpolarisation, tens of seconds are required for complete deactivation at a weakly depolarised potential; this "ultra-sustained activation" is not observed without cAMP. Whilst cAMP slows deactivation of wild-type channels, the K381E mutation amplifies this effect to enable extraordinary kinetic stabilisation of the open state. K381E channels retain S4-gate coupling, with strong voltage dependence of the rate-limiting step for deactivation of mode II channels near -40 mV. At these voltages, the mode I deactivation pathway shows a different rate-limiting step, lacking strong voltage or cAMP dependence. Ultra-sustained activation thus reflects stabilisation of the mode II open state by the K381E mutation in synergistic combination with cAMP binding. Thus, the voltage-sensing domain is subject to strong functional coupling not only to the pore domain but also to the cytoplasmic cAMP-sensing domain in a manner specific to the voltage sensor conformation.

Inflammation-induced increase in hyperpolarization-activated, cyclic nucleotide-gated channel protein in trigeminal ganglion neurons and the effect of buprenorphine.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are active at resting membrane potential and thus contribute to neuronal excitability. Their increased activity has recently been demonstrated in models of nerve injury-induced pain. The major aim of the current study was to investigate altered HCN channel protein expression in trigeminal sensory neurons following inflammation of the dura. HCN1 and HCN2 channel immunoreactivity was observed on the membranes of medium- to large-sized trigeminal ganglion neurons with 76% and 85% of HCN1 and HCN2 expressing neurons also containing the 200 kDa neurofilament protein (associated with myelinated fibers). Western immunoblots of lysates from rat trigeminal ganglia also showed bands with appropriate molecular weights for HCN1 and HCN2. Three days after application of complete Freund's adjuvant (CFA) to the dura mater, Western blot band densities were significantly increased; compared to control, to 166% for HCN1 and 284% for HCN2 channel protein. The band densities were normalized against alpha-actin. In addition, the number of retrogradely labeled neurons from the dura expressing HCN1 and HCN2 was significantly increased to 247% (HCN1) and 171% (HCN2), three days after inflammation. When the opioid receptor partial agonist, buprenorphine, was given systemically, immediately after CFA, the inflammation-induced increase in HCN protein expression in both Western blot and immunohistochemical experiments was not observed. These results suggest that HCN1 and HCN2 are involved in inflammation-induced sensory neuron hyperexcitability, and indicate that an opioid receptor agonist can reverse the protein upregulation.

Cyclic GMP alters the firing rate and thermosensitivity of hypothalamic neurons.

The rostral hypothalamus, especially the preoptic-anterior hypothalamus (POAH), contains temperature-sensitive and -insensitive neurons that form synaptic networks to control thermoregulatory responses. Previous studies suggest that the cyclic nucleotide cGMP is an important mediator in this neuronal network, since hypothalamic microinjections of cGMP analogs produce hypothermia in several species. In the present study, immunohistochemisty showed that rostral hypothalamic neurons contain cGMP, guanylate cyclase (necessary for cGMP synthesis), and CNG A2 (an important cyclic nucleotide-gated channel). Extracellular electrophysiological activity was recorded from different types of neurons in rat hypothalamic tissue slices. Each recorded neuron was classified according to its thermosensitivity as well as its firing rate response to 2-100 microM 8-bromo-cGMP (a membrane-permeable cGMP analog). cGMP has specific effects on different neurons in the rostral hypothalamus. In the POAH, the cGMP analog decreased the spontaneous firing rate in 45% of temperature-sensitive and -insensitive neurons, an effect that is likely due to cGMP-enhanced hyperpolarizing K(+) currents. This decreased POAH activity could attenuate thermoregulatory responses and produce hypothermia during exposures to cool or neutral ambient temperatures. Although 8-bromo-cGMP did not affect the thermosensitivity of most POAH neurons, it did increase the warm sensitivity of neurons in other hypothalamic regions located dorsal, lateral, and posterior to the POAH. This increased thermosensitivity may be due to pacemaker currents that are facilitated by cyclic nucleotides. If some of these non-POAH thermosensitive neurons promote heat loss or inhibit heat production, then their increased thermosensitivity could contribute to cGMP-induced decreases in body temperature.

Preclinical results with I(f) current inhibition by ivabradine.

Ivabradine, a highly selective I(f) current inhibitor acting directly on the sinoatrial node, induces a rapid, sustained and dose-dependent reduction of heart rate at rest and during exercise, without significant effects on atrioventricular conduction, left ventricular (LV) contraction-relaxation or vascular tissues. These properties, associated with an improvement in LV loading related to bradycardia, resulted in an increase in stroke volume and preservation of cardiac output at rest and during exercise. Reducing myocardial oxygen consumption and improving oxygen supply, ivabradine reduced the severity of ischaemia and associated regional contractile dysfunction of the stunned myocardium. Long-term administration of ivabradine in rats with chronic heart failure improved cardiac haemodynamics associated with a progressive remodelling of LV structure. In dyslipidaemic mice, ivabradine prevented the renal and cerebrovascular endothelial dysfunction associated with atherosclerosis. These preclinical data suggest that long-term reduction in heart rate with ivabradine might interact with multiple a priori unexpected mechanisms involved in cardiac and vascular remodelling processes associated with chronic heart diseases.