The Hyperpolarization-Activated HCN4 Channel is Important for Proper Maintenance of Oscillatory Activity in the Thalamocortical System.

Hyperpolarization-activated cation channels are involved, among other functions, in learning and memory, control of synaptic transmission and epileptogenesis. The importance of the HCN1 and HCN2 isoforms for brain function has been demonstrated, while the role of HCN4, the third major neuronal HCN subunit, is not known. Here we show that HCN4 is essential for oscillatory activity in the thalamocortical (TC) network. HCN4 is selectively expressed in various thalamic nuclei, excluding the thalamic reticular nucleus. HCN4-deficient TC neurons revealed a massive reduction of Ih and strongly reduced intrinsic burst firing, whereas the current was normal in cortical pyramidal neurons. In addition, evoked bursting in a thalamic slice preparation was strongly reduced in the mutant mice probes. HCN4-deficiency also significantly slowed down thalamic and cortical oscillations during active wakefulness. Taken together, these results establish that thalamic HCN4 channels are essential for the production of rhythmic intrathalamic oscillations and determine regular TC oscillatory activity during alert states.

Two types of interneurons in the mouse lateral geniculate nucleus are characterized by different h-current density.

Although hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels and the corresponding h-current (Ih) have been shown to fundamentally shape the activity pattern in the thalamocortical network, little is known about their function in local circuit GABAergic interneurons (IN) of the dorsal part of the lateral geniculate nucleus (dLGN). By combining electrophysiological, molecular biological, immunohistochemical and cluster analysis, we characterized the properties of Ih and the expression profile of HCN channels in IN. Passive and active electrophysiological properties of IN differed. Two subclasses of IN were resolved by unsupervised cluster analysis. Small cells were characterized by depolarized resting membrane potentials (RMP), stronger anomalous rectification, higher firing frequency of faster action potentials (APs), appearance of rebound bursting, and higher Ih current density compared to the large IN. The depolarization exerted by sustained HCN channel activity facilitated neuronal firing. In addition to cyclic nucleotides, Ih in IN was modulated by PIP2 probably based on the abundant expression of the HCN3 isoform. Furthermore, only IN with larger cell diameters expressed neuronal nitric oxide synthase (nNOS). It is discussed that Ih in IN is modulated by neurotransmitters present in the thalamus and that the specific properties of Ih in these cells closely reflect their modulatory options.

An N-terminal deletion variant of HCN1 in the epileptic WAG/Rij strain modulates HCN current densities.

Rats of the Wistar Albino Glaxo/Rij (WAG/Rij) strain show symptoms resembling human absence epilepsy. Thalamocortical neurons of WAG/Rij rats are characterized by an increased HCN1 expression, a negative shift in I h activation curve, and an altered responsiveness of I h to cAMP. We cloned HCN1 channels from rat thalamic cDNA libraries of the WAG/Rij strain and found an N-terminal deletion of 37 amino acids. In addition, WAG-HCN1 has a stretch of six amino acids, directly following the deletion, where the wild-type sequence (GNSVCF) is changed to a polyserine motif. These alterations were found solely in thalamus mRNA but not in genomic DNA. The truncated WAG-HCN1 was detected late postnatal in WAG/Rij rats and was not passed on to rats obtained from pairing WAG/Rij and non-epileptic August Copenhagen Irish rats. Heterologous expression in Xenopus oocytes revealed 2.2-fold increased current amplitude of WAG-HCN1 compared to rat HCN1. While WAG-HCN1 channels did not have altered current kinetics or changed regulation by protein kinases, fluorescence imaging revealed a faster and more pronounced surface expression of WAG-HCN1. Using co-expression experiments, we found that WAG-HCN1 channels suppress heteromeric HCN2 and HCN4 currents. Moreover, heteromeric channels of WAG-HCN1 with HCN2 have a reduced cAMP sensitivity. Functional studies revealed that the gain-of-function of WAG-HCN1 is not caused by the N-terminal deletion alone, thus requiring a change of the N-terminal GNSVCF motif. Our findings may help to explain previous observations in neurons of the WAG/Rij strain and indicate that WAG-HCN1 may contribute to the genesis of absence seizures in WAG/Rij rats.

Thalamic Kv 7 channels: pharmacological properties and activity control during noxious signal processing.

BACKGROUND AND PURPOSE: The existence of functional K(v)7 channels in thalamocortical (TC) relay neurons and the effects of the K(+)-current termed M-current (I(M)) on thalamic signal processing have long been debated. Immunocytochemical evidence suggests their presence in this brain region. Therefore, we aimed to verify their existence, pharmacological properties and function in regulating activity in neurons of the ventrobasal thalamus (VB). EXPERIMENTAL APPROACH: Characterization of K(v)7 channels was performed by combining in vitro, in vivo and in silico techniques with a pharmacological approach. Retigabine (30 µM) and XE991 (20 µM), a specific K(v)7 channel enhancer and blocker, respectively, were applied in acute brain slices during electrophysiological recordings. The effects of intrathalamic injection of retigabine (3 mM, 300 nL) and/or XE991 (2 mM, 300 nL) were investigated in freely moving animals during hot-plate tests by recording behaviour and neuronal activity. KEY RESULTS: K(v)7.2 and K(v)7.3 subunits were found to be abundantly expressed in TC neurons of mouse VB. A slow K(+)-current with properties of IM was activated by retigabine and inhibited by XE991. K(v)7 channel activation evoked membrane hyperpolarization, a reduction in tonic action potential firing, and increased burst firing in vitro and in computational models. Single-unit recordings and pharmacological intervention demonstrated a specific burst-firing increase upon I(M) activation in vivo. A K(v)7 channel-mediated increase in pain threshold was associated with fewer VB units responding to noxious stimuli, and increased burst firing in responsive neurons. CONCLUSIONS AND IMPLICATIONS: K(v)7 channel enhancement alters somatosensory activity and may reflect an anti-nociceptive mechanism during acute pain processing.

Regionally specific expression of high-voltage-activated calcium channels in thalamic nuclei of epileptic and non-epileptic rats.

The polygenic origin of generalized absence epilepsy results in dysfunction of ion channels that allows the switch from physiological asynchronous to pathophysiological highly synchronous network activity. Evidence from rat and mouse models of absence epilepsy indicates that altered Ca(2+) channel activity contributes to cellular and network alterations that lead to seizure activity. Under physiological circumstances, high voltage-activated (HVA) Ca(2+) channels are important in determining the thalamic firing profile. Here, we investigated a possible contribution of HVA channels to the epileptic phenotype using a rodent genetic model of absence epilepsy. In this study, HVA Ca(2+) currents were recorded from neurons of three different thalamic nuclei that are involved in both sensory signal transmission and rhythmic-synchronized activity during epileptic spike-and-wave discharges (SWD), namely the dorsal part of the lateral geniculate nucleus (dLGN), the ventrobasal thalamic complex (VB) and the reticular thalamic nucleus (NRT) of epileptic Wistar Albino Glaxo rats from Rijswijk (WAG/Rij) and non-epileptic August Copenhagen Irish (ACI) rats. HVA Ca(2+) current densities in dLGN neurons were significantly increased in epileptic rats compared with non-epileptic controls while other thalamic regions revealed no differences between the strains. Application of specific channel blockers revealed that the increased current was carried by L-type Ca(2+) channels. Electrophysiological evidence of increased L-type current correlated with up-regulated mRNA and protein expression of a particular L-type channel, namely Cav1.3, in dLGN of epileptic rats. No significant changes were found for other HVA Ca(2+) channels. Moreover, pharmacological inactivation of L-type Ca(2+) channels results in altered firing profiles of thalamocortical relay (TC) neurons from non-epileptic rather than from epileptic rats. While HVA Ca(2+) channels influence tonic and burst firing in ACI and WAG/Rij differently, it is discussed that increased Cav1.3 expression may indirectly contribute to increased robustness of burst firing and thereby the epileptic phenotype of absence epilepsy.

Thalamic involvement in patients with neurologic impairment due to Shiga toxin 2.

OBJECTIVE: The outbreak of hemolytic-uremic syndrome and diarrhea caused by Shiga toxin-producing Escherichia coli O104:H4 in Germany during May to July 2011 involved severe and characteristic neurologic manifestations with a strong female preponderance. Owing to these observations, we designed a series of experimental studies to evaluate the underlying mechanism of action of this clinical picture. METHODS: A magnetic resonance imaging and electroencephalographic study of patients was performed to evaluate the clinical picture in detail. Thereafter, combinations of different experimental settings, including electrophysiological and histological analyses, as well as calcium imaging in brain slices of rats, were conducted. RESULTS: We report on 7 female patients with neurologic symptoms and signs including bilateral thalamic lesions and encephalopathic changes indicative of a predominant involvement of the thalamus. Experimental studies in rats revealed an enhanced expression of the Shiga toxin receptor globotriaosylceramide on thalamic neurons in female rats as compared to other brain regions in the same rats and to male animals. Incubation of brain slices with Shiga toxin 2 evoked a strong membrane depolarization and intracellular calcium accumulation in neurons, associated with neuronal apoptosis, predominantly in the thalamic area. INTERPRETATION: These findings suggest that the direct cytotoxic effect of Shiga toxin 2 in the thalamus might contribute to the pathophysiology of neuronal complications in hemolytic-uremic syndrome.

Ca(2+)-dependent large conductance K(+) currents in thalamocortical relay neurons of different rat strains.

Mutations in genes coding for Ca(2+) channels were found in patients with childhood absence epilepsy (CAE) indicating a contribution of Ca(2+)-dependent mechanisms to the generation of spike-wave discharges (SWD) in humans. Since the involvement of Ca(2+) signals remains unclear, the aim of the present study was to elucidate the function of a Ca(2+)-dependent K(+) channel (BKCa) under physiological conditions and in the pathophysiological state of CAE. The activation of BKCa channels is dependent on both voltage and intracellular Ca(2+) concentrations. Moreover, these channels exhibit an outstandingly high level of regulatory heterogeneity that builds the basis for the influence of BKCa channels on different aspects of neuronal activity. Here, we analyse the contribution of BKCa channels to firing of thalamocortical relay neurons, and we test the hypothesis that BKCa channel activity affects the phenotype of a genetic rat model of CAE. We found that the activation of the ß2-adrenergic receptor/protein kinase A pathway resulted in BKCa channel inhibition. Furthermore, BKCa channels affect the number of action potentials fired in a burst and produced spike frequency adaptation during tonic activity. The latter result was confirmed by a computer modelling approach. We demonstrate that the ß2-adrenergic inhibition of BKCa channels prevents spike frequency adaptation and, thus, might significantly support the tonic firing mode of thalamocortical relay neurons. In addition, we show that BKCa channel functioning differs in epileptic WAG/Rij and thereby likely contributes to highly synchronised, epileptic network activity.

Adenylyl cyclases: expression in the developing rat thalamus and their role in absence epilepsy.

Adenylyl cyclases (ACs) synthesize the second messenger cyclic AMP (cAMP) which influences the function of multiple ion channels. Former studies point to a malfunction of cAMP-dependent ion channel regulation in thalamocortical relay neurons that contribute to the development of the absence epileptic phenotype of a rat genetic model (WAG/Rij). Here, we provide detailed information about the thalamic gene and protein expression of Ca(2+)/calmodulin-activated AC isoforms in rat thalamus. Data from WAG/Rij were compared to those from non-epileptic controls (August-Copenhagen Irish rats) to elucidate whether differential expression of ACs contributes to the dysregulation of thalamocortical activity. At one postnatal stage (P21), we found the gene expression of two specific Ca(2+)-activated AC isoforms (AC-1 and AC-3) to be significantly down-regulated in epileptic tissue, and we identified the isoform AC-1 to be the most prominent one in both strains. However, Western blot data and analysis of enzymatic AC activity revealed no differences between the two strains. While basal AC activity was low, cAMP production was boosted by application of a forskolin derivative up to sevenfold. Despite previous hints pointing to a major contribution of ACs, the presented data show that there is no apparent causality between AC activity and the occurrence of the epileptic phenotype.

Identification of the muscarinic pathway underlying cessation of sleep-related burst activity in rat thalamocortical relay neurons.

Modulation of the standing outward current (I (SO)) by muscarinic acetylcholine (ACh) receptor (MAChR) stimulation is fundamental for the state-dependent change in activity mode of thalamocortical relay (TC) neurons. Here, we probe the contribution of MAChR subtypes, G proteins, phospholipase C (PLC), and two pore domain K(+) (K(2P)) channels to this signaling cascade. By the use of spadin and A293 as specific blockers, we identify TWIK-related K(+) (TREK)-1 channel as new targets and confirm TWIK-related acid-sensitve K(+) (TASK)-1 channels as known effectors of muscarinic signaling in TC neurons. These findings were confirmed using a high affinity blocker of TASK-3 and TREK-1, namely, tetrahexylammonium chloride. It was found that the effect of muscarinic stimulation was inhibited by M(1)AChR-(pirenzepine, MT-7) and M(3)AChR-specific (4-DAMP) antagonists, phosphoinositide-specific PLCß (PI-PLC) inhibitors (U73122, ET-18-OCH(3)), but not the phosphatidylcholine-specific PLC (PC-PLC) blocker D609. By comparison, depleting guanosine-5'-triphosphate (GTP) in the intracellular milieu nearly completely abolished the effect of MAChR stimulation. The block of TASK and TREK channels was accompanied by a reduction of the muscarinic effect on I (SO). Current-clamp recordings revealed a membrane depolarization following MAChR stimulation, which was sufficient to switch TC neurons from burst to tonic firing under control conditions but not during block of M(1)AChR/M(3)AChR and in the absence of intracellular GTP. These findings point to a critical role of G proteins and PLC as well as TASK and TREK channels in the muscarinic modulation of thalamic activity modes.

Differential regulation of HCN channel isoform expression in thalamic neurons of epileptic and non-epileptic rat strains.

Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels represent the molecular substrate of the hyperpolarization-activated inward current (I(h)). Although these channels act as pacemakers for the generation of rhythmic activity in the thalamocortical network during sleep and epilepsy, their developmental profile in the thalamus is not yet fully understood. Here we combined electrophysiological, immunohistochemical, and mathematical modeling techniques to examine HCN gene expression and I(h) properties in thalamocortical relay (TC) neurons of the dorsal part of the lateral geniculate nucleus (dLGN) in an epileptic (WAG/Rij) compared to a non-epileptic (ACI) rat strain. Recordings of TC neurons between postnatal day (P) 7 and P90 in both rat strains revealed that I(h) was characterized by higher current density, more hyperpolarized voltage dependence, faster activation kinetics, and reduced cAMP-sensitivity in epileptic animals. All four HCN channel isoforms (HCN1-4) were detected in dLGN, and quantitative analyses revealed a developmental increase of protein expression of HCN1, HCN2, and HCN4 but a decrease of HCN3. HCN1 was expressed at higher levels in WAG/Rij rats, a finding that was correlated with increased expression of the interacting proteins filamin A (FilA) and tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b). Analysis of a simplified computer model of the thalamic network revealed that the alterations of I(h) found in WAG/Rij rats compensate each other in a way that leaves I(h) availability constant, an effect that ensures unaltered cellular burst activity and thalamic oscillations. These data indicate that during postnatal developmental the hyperpolarizing shift in voltage dependency (resulting in less current availability) is compensated by an increase in current density in WAG/Rij thereby possibly limiting the impact of I(h) on epileptogenesis. Because HCN3 is expressed higher in young versus older animals, HCN3 likely does not contribute to alterations in I(h) in older animals.

Modulation of calcium-dependent inactivation of L-type Ca2+ channels via ß-adrenergic signaling in thalamocortical relay neurons.

Neuronal high-voltage-activated (HVA) Ca(2+) channels are rapidly inactivated by a mechanism that is termed Ca(2+)-dependent inactivation (CDI). In this study we have shown that ß-adrenergic receptor (ßAR) stimulation inhibits CDI in rat thalamocortical (TC) relay neurons. This effect can be blocked by inhibition of cAMP-dependent protein kinase (PKA) with a cell-permeable inhibitor (myristoylated protein kinase inhibitor-(14-22)-amide) or A-kinase anchor protein (AKAP) St-Ht31 inhibitory peptide, suggesting a critical role of these molecules downstream of the receptor. Moreover, inhibition of protein phosphatases (PP) with okadaic acid revealed the involvement of phosphorylation events in modulation of CDI after ßAR stimulation. Double fluorescence immunocytochemistry and pull down experiments further support the idea that modulation of CDI in TC neurons via ßAR stimulation requires a protein complex consisting of Ca(V)1.2, PKA and proteins from the AKAP family. All together our data suggest that AKAPs mediate targeting of PKA to L-type Ca(2+) channels allowing their phosphorylation and thereby modulation of CDI.

A-type K+ currents in intralaminar thalamocortical relay neurons.

Transient A-type K+ currents (I(A)) are known to influence the firing pattern of a number of thalamic cell types, but have not been investigated in intralaminar thalamocortical (TC) relay neurons yet. We therefore combined whole-cell patch-clamp techniques, PCR analysis, and immunohistochemistry to investigate the voltage-dependent and pharmacological properties of I(A) and to determine its molecular basis in TC neurons from the centrolateral, paracentral, and centromedial thalamic nuclei. I(A) revealed half-maximal (V (h)) activation and inactivation at about -17 and -67 mV, respectively. At a concentration of 5-10 mM 4-aminopyridine (4-AP) completely blocked I(A). Furthermore, I(A) was nearly unaffected by two sea anemone toxins (blood depressing substances 1 and 2, BDS1 and BDS2; 6-8% block at a concentration of 1 µM) but strongly sensitive to the K(V)4 channel blocker Heteropoda venatoria toxin 2 (HpTx2; about 45% block at a concentration of 5 µM). PCR screening revealed the expression of K(V)4.1-4.3, with strongest expression for K(V)4.2 and weak expression for K(V)4.1. Accordingly K(V)4.1 was not detected in immunohistochemical staining. Furthermore, K(V)4.2 and K(V)4.3 revealed mainly dendritic and somatic staining, respectively. Together with current clamp recordings, these findings point to a scenario where the fast transient I(A) in intralaminar TC neurons has a depolarized threshold at potentials negative to -50 mV, is substantially generated by K(V)4.2 and K(V)4.3 channels, allows prominent burst firing at hyperpolarized potentials, prevents the generation of high-threshold potentials, generates a delayed onset of firing at more depolarized potentials, and allows fast tonic firing.

Activity Modes in Thalamocortical Relay Neurons are Modulated by G(q)/G(11) Family G-proteins - Serotonergic and Glutamatergic Signaling.

In thalamocortical relay (TC) neurons, G-protein-coupled receptors play an important part in the control of activity modes. A conditional Gα(q) knockout on the background of a constitutive Gα(11) knockout (Gα(q)/Gα(11) (-/-)) was used to determine the contribution of Gq/G11 family G-proteins to metabotropic serotonin (5-HT) and glutamate (Glu) function in the dorsal part of the lateral geniculate nucleus (dLGN). In control mice, current clamp recordings showed that α-m-5-HT induced a depolarization of V(rest) which was sufficient to suppress burst firing. This depolarization was concentration-dependent (100 µM: +6 ± 1 mV, n = 10; 200 µM: +10 ± 1 mV, n = 7) and had a conditioning effect on the activation of other Gα(q)-mediated pathways. The depolarization was significantly reduced in Gα(q)/Gα(11) (-/-) (100 µM: 3 ± 1 mV, n = 11; 200 µM: 5 ± 1 mV, n = 6) and was apparently insufficient to suppress burst firing. Activating Gα(q)-coupled muscarinic receptors affected the magnitude of α-m-5-HT-induced effects in a reciprocal manner. Furthermore, the depolarizing effect of mGluR1 agonists was significantly reduced in Gα(q)/Gα(11) (-/-) mice. Immunohistochemical stainings revealed binding of 5-HT(2C)R- and mGluR1α-, but not of 5-HT(2A)R-specific antibodies in the dLGN of Gα(q)/Gα(11) (-/-) mice. In conclusion, these findings demonstrate that transmitters of ascending brainstem fibers and corticofugal fibers both signal via a central element in the form of Gq/G11-mediated pathways to control activity modes in the TC system.

Burst discharges in neurons of the thalamic reticular nucleus are shaped by calcium-induced calcium release.

The nucleus reticularis thalami (NRT) is a layer of inhibitory neurons that surrounds the dorsal thalamus. It appears to be the 'pacemaker' of certain forms of slow oscillations in the thalamus and was proposed to be a key determinant of the internal attentional searchlight as well as the origin of hypersynchronous activity during absence seizures. Neurons of the NRT exhibit a transient depolarization termed low threshold spike (LTS) following sustained hyperpolarization. This is caused by the activation of low-voltage-activated Ca2+ channels (LVACC). Although the role of these channels in thalamocortical oscillations was studied in great detail, little is known about the downstream intracellular Ca2+ signalling pathways and their feedback onto the oscillations. A signalling triad consisting of the sarco(endo)plasmic reticulum calcium ATPase (SERCA), Ca2+ activated K+ channels (SK2), and LVACC is active in dendrites of NRT neurons and shapes rhythmic oscillations. The aim of our study was to find out (i) if and how Ca2+-induced Ca2+ release (CICR) via ryanodine receptors (RyR) can be evoked in NRT neurons and (ii) how the released Ca2+ affects burst activity. Combining electrophysiological, immunohistochemical, and two-photon Ca2+ imaging techniques, we show that CICR in NRT neurons takes place by a cell-type specific coupling of LVACC and RyR. CICR could be evoked by the application of caffeine, by activation of LVACC, or by repetitive LTS generation. During the latter, CICR contributed 30% to the resulting build-up of [Ca2+]i. CICR was abolished by cyclopiazonic acid, a specific blocker for SERCA, or by high concentrations of ryanodine (50 microM). Unlike other thalamic nuclei, in the NRT the activation of high-voltage-activated Ca2+ channels failed to evoke CICR. While action potentials contributed little to the build-up of [Ca2+]i upon repetitive LTS generation, the Ca2+ released via RyR significantly reduced the number of action potentials during an LTS and reduced the neurons' low threshold activity, thus potentially reducing hypersynchronicity. This effect persisted in the presence of the SK2 channel blocker apamin. We conclude that the activation of LVACC specifically causes CICR via RyR in neurons of the NRT, thereby adding a Ca2+-dependent intracellular route to the mechanisms determining rhythmic oscillatory bursting in this nucleus.

Postnatal expression pattern of HCN channel isoforms in thalamic neurons: relationship to maturation of thalamocortical oscillations.

Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels are the molecular substrate of the hyperpolarization-activated inward current (I(h)). Because the developmental profile of HCN channels in the thalamus is not well understood, we combined electrophysiological, molecular, immunohistochemical, EEG recordings in vivo, and computer modeling techniques to examine HCN gene expression and I(h) properties in rat thalamocortical relay (TC) neurons in the dorsal part of the lateral geniculate nucleus and the functional consequence of this maturation. Recordings of TC neurons revealed an approximate sixfold increase in I(h) density between postnatal day 3 (P3) and P106, which was accompanied by significantly altered current kinetics, cAMP sensitivity, and steady-state activation properties. Quantification on tissue levels revealed a significant developmental decrease in cAMP. Consequently the block of basal adenylyl cyclase activity was accompanied by a hyperpolarizing shift of the I(h) activation curve in young but not adult rats. Quantitative analyses of HCN channel isoforms revealed a steady increase of mRNA and protein expression levels of HCN1, HCN2, and HCN4 with reduced relative abundance of HCN4. Computer modeling in a simplified thalamic network indicated that the occurrence of rhythmic delta activity, which was present in the EEG at P12, differentially depended on I(h) conductance and modulation by cAMP at different developmental states. These data indicate that the developmental increase in I(h) density results from increased expression of three HCN channel isoforms and that isoform composition and intracellular cAMP levels interact in determining I(h) properties to enable progressive maturation of rhythmic slow-wave sleep activity patterns.

Correlation of T-channel coding gene expression, IT, and the low threshold Ca2+ spike in the thalamus of a rat model of absence epilepsy.

T-type Ca(2+) current-dependent burst firing of thalamic neurons is thought to be involved in the hyper-synchronous activity observed during absence seizures. Here we investigate the correlation between the expression of T-channel coding genes (alpha1G, -H, -I), T-type Ca(2+) current, and the T-current-dependent low threshold Ca(2+) spike in three functionally distinct thalamic nuclei (lateral geniculate nucleus; centrolateral nucleus; reticular nucleus) in a rat model of absence epilepsy, the WAG/Rij rats, and a non-epileptic control strain, the ACI rats. The lateral geniculate nucleus and centrolateral nucleus were found to primarily express alpha1G and alpha1I, while the reticular thalamic nucleus expressed alpha1H and alpha1I. Expression was higher in WAG/Rij when compared to ACI. The T-type Ca(2+) current properties matched the predictions derived from the expression pattern analysis. Current density was larger in all nuclei of WAG/Rij rats when compared to ACI and correlated with LTS size and the minimum LTS generating slope, while T-type Ca(2+) current voltage dependency correlated with the LTS onset potential.

Reciprocal modulation of I (h) and I (TASK) in thalamocortical relay neurons by halothane.

By combining electrophysiological, immunohistochemical, and computer modeling techniques, we examined the effects of halothane on the standing outward current (I (SO)) and the hyperpolarization-activated current (I (h)) in rat thalamocortical relay (TC) neurons of the dorsal lateral geniculate nucleus (dLGN). Hyperpolarizing voltage steps elicited an instantaneous current component (I (i)) followed by a slower time-dependent current that represented I (h). Halothane reduced I (h) by shifting the voltage dependency of activation toward more negative potentials and by reducing the maximal conductance. Moreover, halothane augmented I (i) and I (SO). During the blockade of I (h) through Cs+, the current-voltage relationship of the halothane-sensitive current closely resembled the properties of a current through members of the TWIK-related acid-sensitive K+ (TASK) channel family (I (TASK)). Computer simulations in a single-compartment TC neuron model demonstrated that the modulation of I (h) and I (TASK) is sufficient to explain the halothane-induced hyperpolarization of the membrane potential observed in current clamp recordings. Immunohistochemical staining revealed protein expression of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel proteins HCN1, HCN2, and HCN4. Together with the dual effect of halothane on I (h) properties, these results suggest that I (h) in TC neurons critically depends on HCN1/HCN2 heterodimers. It is concluded that the reciprocal modulation of I (h) and I (TASK) is an important mechanism of halothane action in the thalamus.

Muscarinic ACh receptor-mediated control of thalamic activity via G(q)/G (11)-family G-proteins.

A genetic knock out was used to determine the specific contribution of G(q)/G(11)-family G-proteins to the function of thalamocortical relay (TC) neurons. Disruption of Galpha(q) function in a conditional forebrain-specific Galpha(q)/Galpha(11)-double-deficient mouse line (Galpha(q)/Galpha(11)(-/-) had no effects on the resting membrane potential (V (rest)) and the amplitude of the standing outward current (I (SO)). Stimulation of muscarinic acetylcholine (ACh) receptors (mAChR; muscarine, 50 microM) induced a decrease in I (SO) amplitude in wild-type mice (36 +/- 4%, n = 5), a constitutive Galpha(11)-deficient mouse line (Galpha(11)(-/-; 36 +/- 3%, n = 8), and Galpha(q)/Galpha(11)(-/-) (11 +/- 2%, n = 16). Current-clamp recordings revealed a muscarine-induced positive shift in V (rest) of 23 +/- 2 mV (n = 6), 18 +/- 5 mV (n = 5), and 2 +/- 1 mV (n = 9) in wild type, Galpha(11)(-/-), and Galpha(q)/Galpha(11)(-/-), respectively. This depolarization was associated with a change in TC neuron activity from burst to tonic firing in wild type and Galpha(11)(-/-), but not in Galpha(q)/Galpha(11)(-/-). The use of specific antibodies and of pharmacological agents with preferred affinity points to the contribution of m(1)AChR and m(3)AChR. In conclusion, we present two novel aspects of the physiology of the thalamocortical system by demonstrating that the depolarization of TC neurons, which is induced by the action of transmitters of ascending brainstem fibers, is governed roughly equally by both m(1)AChR and m(3)AChR and is transduced by Galpha(q) but not by Galpha(11).

The two-pore domain potassium channel TASK3 functionally impacts glioma cell death.

Two-pore domain K(+) channels, a recently discovered family of ion channels with a unique membrane topology, have been shown to be critically involved in cell death. We here address the functional role of TASK3 (TWIK-related acid-sensitive K(+) channel, KCNK9) in human glioblastoma in vitro and in vivo. Human glioma cell lines (n = 5) as well as glioma specimens (n = 5) constitutively express TASK3 mRNA and protein. The functional impact of the potassium channel on cell survival was investigated using a medium with high (25 mM) extracellular potassium over 7 days. Using flow cytometric assessment, we show that under these culture conditions 97 +/- 0.76% of all glioma cells survived. Application of the TASK channel opener isoflurane (1 vol%) resulted in a 30 +/- 4% reduction of cell survival in different glioma cell lines. Simultaneous application of isoflurane and the TASK channel blockers bupivacaine (20 microM) and spermine (500 microM) completely reversed this effect. Our results demonstrate the expression of TASK3 in glioma cells in vitro and in vivo and provide a direct link between the TASK3 channel function and glioma cell survival. This implies that TASK3 channels may possibly represent a novel molecular target for the treatment of this type of cancer.

T-current related effects of antiepileptic drugs and a Ca2+ channel antagonist on thalamic relay and local circuit interneurons in a rat model of absence epilepsy.

Channel blocking, anti-oscillatory, and anti-epileptic effects of clinically used anti-absence substances (ethosuximide, valproate) and the T-type Ca2+ current (IT) blocker mibefradil were tested by analyzing membrane currents in acutely isolated local circuit interneurons and thalamocortical relay (TC) neurons, slow intrathalamic oscillations in brain slices, and spike and wave discharges (SWDs) occurring in vivo in Wistar Albino Glaxo rats from Rijswijk (WAG/Rij). Substance effects in vitro were compared between WAG/Rij and a non-epileptic control strain, the ACI rats. Ethosuximide (ETX) and valproate were found to block IT in acutely isolated thalamic neurons. Block of IT by therapeutically relevant ETX concentrations (0.25-0.75 mM) was stronger in WAG/Rij, although the maximal effect at saturating concentrations (>or=10 mM) was stronger in ACI. Ethosuximide delayed the onset of the low threshold Ca2+ spike (LTS) of neurons recorded in slice preparations. Mibefradil (>or=2 microM) completely blocked IT and the LTS, dampened evoked thalamic oscillations, and attenuated SWDs in vivo. Computational modeling demonstrated that the complete effect of ETX can be replicated by a sole reduction of IT. However, the necessary degree of IT reduction was not induced by therapeutically relevant ETX concentrations. A combined reduction of IT, the persistent sodium current, and the Ca2+ activated K+ current resulted in an LTS alteration resembling the experimental observations. In summary, these results support the hypothesis of IT reduction as part of the mechanism of action of anti-absence drugs and demonstrate the ability of a specific IT antagonist to attenuate rhythmic burst firing and SWDs.