Manuela Cerina

Differential phospholipase C-dependent modulation of TASK and TREK two-pore domain K+ channels in rat thalamocortical relay neurons

During the behavioural states of sleep and wakefulness thalamocortical relay neurons fire action potentials in high frequency bursts or tonic sequences, respectively. The modulation of specific K+ channel types, termed TASK and TREK, allows these neurons to switch between the two modes of activity. In this study we show that the signalling lipids phosphatidylinositol 4,5‐bisphosphate (PIP2) and diacylglycerol (DAG), which are components of their membrane environment, switch on and shut off TREK and TASK channels, respectively. These channel modulations contribute to a better understanding of the molecular basis of the effects of neurotransmitters such as ACh which are released by the brainstem arousal system. The present report introduces PIP2 and DAG as new elements of signal transduction in the thalamus.

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

Modulation of the standing outward current (ISO) 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+ (K2P) 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 M1AChR-(pirenzepine, MT-7) and M3AChR-specific (4-DAMP) antagonists, phosphoinositide-specific PLCβ (PI-PLC) inhibitors (U73122, ET-18-OCH3), 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 ISO. 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 M1AChR/M3AChR 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.

The role of two-pore-domain background K+ (K2P) channels in the thalamus

The thalamocortical system is characterized by two fundamentally different activity states, namely synchronized burst firing and tonic action potential generation, which mainly occur during the behavioral states of sleep and wakefulness, respectively. The switch between the two firing modes is crucially governed by the bidirectional modulation of members of the K2P channel family, namely tandem of P domains in a weakly inward rectifying K+ (TWIK)-related acid-sensitive K+ (TASK) and TWIK-related K+ (TREK) channels, in thalamocortical relay (TC) neurons. Several physicochemical stimuli including neurotransmitters, protons, di- and multivalent cations as well as clinically used drugs have been shown to modulate K2P channels in these cells. With respect to modulation of these channels by G-protein-coupled receptors, PLCβ plays a unique role with both substrate breakdown and product synthesis exerting important functions. While the degradation of PIP2 leads to the closure of TREK channels, the production of DAG induces the inhibition of TASK channels. Therefore, TASK and TREK channels were found to be central elements in the control of thalamic activity modes. Since research has yet focused on identifying the muscarinic pathway underling the modulation of TASK and TREK channels in TC neurons, future studies should address other thalamic cell types and members of the K2P channel family.

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

Mutations in genes coding for Ca2+ channels were found in patients with childhood absence epilepsy (CAE) indicating a contribution of Ca2+-dependent mechanisms to the generation of spike-wave discharges (SWD) in humans. Since the involvement of Ca2+ signals remains unclear, the aim of the present study was to elucidate the function of a Ca2+-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 Ca2+ 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 β2-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 β2-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.

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.

Thalamic Kv7 channels: pharmacological properties and activity control during noxious signal processing

The existence of functional Kv7 channels in thalamocortical (TC) relay neurons and the effects of the K+‐current termed M‐current (IM) 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).

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.

The quality of cortical network function recovery depends on localization and degree of axonal demyelination.

Myelin loss is a severe pathological hallmark common to a number of neurodegenerative diseases, including multiple sclerosis (MS). Demyelination in the central nervous system appears in the form of lesions affecting both white and gray matter structures. The functional consequences of demyelination on neuronal network and brain function are not well understood. Current therapeutic strategies for ameliorating the course of such diseases usually focus on promoting remyelination, but the effectiveness of these approaches strongly depends on the timing in relation to the disease state. In this study, we sought to characterize the time course of sensory and behavioral alterations induced by de- and remyelination to establish a rational for the use of remyelination strategies. By taking advantage of animal models of general and focal demyelination, we tested the consequences of myelin loss on the functionality of the auditory thalamocortical system: a well-studied neuronal network consisting of both white and gray matter regions. We found that general demyelination was associated with a permanent loss of the tonotopic cortical organization in vivo, and the inability to induce tone-frequency-dependent conditioned behaviors, a status persisting after remyelination. Targeted, focal lysolecithin-induced lesions in the white matter fiber tract, but not in the gray matter regions of cortex, were fully reversible at the morphological, functional and behavioral level. These findings indicate that remyelination of white and gray matter lesions have a different functional regeneration potential, with the white matter being able to regain full functionality while cortical gray matter lesions suffer from permanently altered network function. Therefore therapeutic interventions aiming for remyelination have to consider both region- and time-dependent strategies.

Chemokine CCL17 is expressed by dendritic cells in the CNS during experimental autoimmune encephalomyelitis and promotes pathogenesis of disease

The CC chemokine ligand 17 (CCL17) and its cognate CC chemokine receptor 4 (CCR4) are known to control leukocyte migration, maintenance of TH17 cells, and regulatory T cell (Treg) expansion in vivo. In this study we characterized the expression and functional role of CCL17 in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). Using a CCL17/EGFP reporter mouse model, we could show that CCL17 expression in the CNS can be found in a subset of classical dendritic cells (DCs) that immigrate into the CNS during the effector phase of MOG-induced EAE. CCL17 deficient (CCL17-/-) mice exhibited an ameliorated disease course upon MOG-immunization, associated with reduced immigration of IL-17 producing CD4+ T cells and peripheral DCs into the CNS. CCL17-/- DCs further showed equivalent MHC class II and costimulatory molecule expression and an equivalent capacity to secrete IL-23 and induce myelin-reactive TH17 cells when compared to wildtype DCs. In contrast, their transmigration in an in vitro model of the blood-brain barrier was markedly impaired. In addition, peripheral Treg cells were enhanced in CCL17-/- mice at peak of disease pointing towards an immunoregulatory function of CCL17 in EAE. Our study identifies CCL17 as a unique modulator of EAE pathogenesis regulating DC trafficking as well as peripheral Treg cell expansion in EAE. Thus, CCL17 operates at distinct levels and on different cell subsets during immune response in EAE, a property harboring therapeutic potential for the treatment of CNS autoimmunity.

Thalamocortical-auditory network alterations following cuprizone‐induced demyelination

BackgroundDemyelination and remyelination are common pathological processes in many neurological disorders, including multiple sclerosis (MS). Clinical evidence suggests extensive involvement of the thalamocortical (TC) system in patients suffering from MS.MethodsUsing murine brain slices of the primary auditory cortex, we investigated the functional consequences of cuprizone-induced de- and remyelination on neuronal activity and auditory TC synaptic transmission in vitro.ResultsOur results revealed an impact of myelin loss and restoration on intrinsic cellular firing patterns, synaptic transmission, and neuronal plasticity in layer 3 and 4 neurons of the auditory TC network. While there was a complex hyper- and depolarizing shift of the resting membrane potential, spontaneous and induced action potential firing was reduced during demyelination and early remyelination. In addition, excitatory postsynaptic potential amplitudes were decreased and induction of LTP was reduced during demyelination.ConclusionsThese data indicate that demyelination-induced impairment of neurons and network activity within the TC system may underlie clinical symptoms observed in demyelinating diseases, corroborating human findings that disease progression is significantly correlated with microstructural tissue damage of the TC system. Further investigation into focal inflammation-induced demyelination models ex vivo and in vivo are needed to understand the functional implication of local and remote lesion formation on TC network activity in MS.