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.

Key pointsr During the behavioural states of sleep and wakefulness thalamocortical relay neurons fire action potentials in high frequency bursts or tonic sequences, respectively.r The modulation of specific K + channel types, termed TASK and TREK, allows these neurons to switch between the two modes of activity.r In this study we show that the signalling lipids phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and diacylglycerol (DAG), which are components of their membrane environment, switch on and shut off TREK and TASK channels, respectively. r 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. r The present report introduces PIP 2 and DAG as new elements of signal transduction in the thalamus. AbstractThe activity of two-pore domain potassium channels (K 2P ) regulates the excitability and firing modes of thalamocortical (TC) neurons. In particular, the inhibition of two-pore domain weakly inwardly rectifying K + channel (TWIK)-related acid-sensitive K + (TASK) channels and TWIK-related K + (TREK) channels, as a consequence of the stimulation of muscarinic ACh receptors (MAChRs) which are coupled to phosphoinositide-specific phospholipase C (PLCβ), induces a shift from burst to tonic firing. By using a whole cell patch-clamp approach, the contribution of the membrane-bound second messenger molecules phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and diacylglycerol (DAG) acting downstream of PLCβ was probed. The standing outward current (I SO ) was used to monitor the current through TASK and TREK channels in TC neurons. By exploiting different manoeuvres to change the intracellular PIP 2 level in TC neurons, we here show that the scavenging of PIP 2 (by neomycin) results in an increased muscarinic effect on I SO whereas increased availability of PIP 2 (inclusion to the patch pipette; histone-based carrier) decreased muscarinic signalling. The degree of muscarinic inhibition specifically depends on phosphatidylinositol phosphate (PIP) and PIP 2 but no other S. G. Meuth and T. Budde contributed equally to this work. phospholipids (phosphatidic acid, phosphatidylserine). The use of specific blockers revealed that PIP 2 is targeting TREK but not TASK channels. Furthermore, we demonstrate that the inhibition of TASK channels is induced by the application of the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG). Under current clamp conditions the activation of MAChRs and PLCβ as well as the application of OAG resulted in membrane depolarization, while PIP 2 application via histone carrier induced a hyperpolarization. These results demonstrate a differential role of PIP 2 and DAG in K 2P channel modulation in native neurons which allows a fine-tuned inhibition of TREK (via PIP 2 depletion) and TASK (via DAG) channels following MAChR stimulation.

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.

The dorsal part of the lateral geniculate nucleus (dLGN) is the main thalamic site for state-dependent transmission of visual information. Non-retinal inputs from the ascending arousal system and inhibition provided by γ-aminobutyric acid (GABA)ergic local circuit interneurons (INs) control neuronal activity within the dLGN. In particular, acetylcholine (ACh) depolarizes thalamocortical relay neurons by inhibiting two-pore domain potassium (K ) channels. Conversely, ACh also hyperpolarizes INs via an as-yet-unknown mechanism. By using whole cell patch-clamp recordings in brain slices and appropriate pharmacological tools we here report that stimulation of type 2 muscarinic ACh receptors induces IN hyperpolarization by recruiting the G-protein βγ subunit (Gβγ), class-1A phosphatidylinositol-4,5-bisphosphate 3-kinase, and cellular and sarcoma (c-Src) tyrosine kinase, leading to activation of two-pore domain weakly inwardly rectifying K channel (TWIK)-related acid-sensitive K (TASK)-1 channels. The latter was confirmed by the use of TASK-1-deficient mice. Furthermore inhibition of phospholipase Cβ as well as an increase in the intracellular level of phosphatidylinositol-3,4,5-trisphosphate facilitated the muscarinic effect. Our results have uncovered a previously unknown role of c-Src tyrosine kinase in regulating IN function in the brain and identified a novel mechanism by which TASK-1 channels are activated in neurons.