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.

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 I_h 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.

Key points: 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 PIP₂and DAG as new elements of signal transduction in the thalamus. The 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 (PIP₂) 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 PIP₂level in TC neurons, we here show that the scavenging of PIP₂(by neomycin) results in an increased muscarinic effect onI_SOwhereas increased availability of PIP₂(inclusion to the patch pipette; histone‐based carrier) decreased muscarinic signalling. The degree of muscarinic inhibition specifically depends on phosphatidylinositol phosphate (PIP) and PIP₂but no other phospholipids (phosphatidic acid, phosphatidylserine). The use of specific blockers revealed that PIP₂is 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 PIP₂application via histone carrier induced a hyperpolarization. These results demonstrate a differential role of PIP₂and DAG in K_2Pchannel modulation in native neurons which allows a fine‐tuned inhibition of TREK (via PIP₂depletion) and TASK (via DAG) channels following MAChR stimulation.

Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels have important functions in controlling neuronal excitability and generating rhythmic oscillatory activity. The role of tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) in regulation of hyperpolarization-activated inward current, I _h, in the thalamocortical system and its functional relevance for the physiological thalamocortical oscillations were investigated. A significant decrease in I _h current density, in both thalamocortical relay (TC) and cortical pyramidal neurons was found in TRIP8b-deficient mice (TRIP8b^−/−). In addition basal cAMP levels in the brain were found to be decreased while the availability of the fast transient A-type K⁺ current, I _A, in TC neurons was increased. These changes were associated with alterations in intrinsic properties and firing patterns of TC neurons, as well as intrathalamic and thalamocortical network oscillations, revealing a significant increase in slow oscillations in the delta frequency range (0.5–4 Hz) during episodes of active-wakefulness. In addition, absence of TRIP8b suppresses the normal desynchronization response of the EEG during the switch from slow-wave sleep to wakefulness. It is concluded that TRIP8b is necessary for the modulation of physiological thalamocortical oscillations due to its direct effect on HCN channel expression in thalamus and cortex and that mechanisms related to reduced cAMP signaling may contribute to the present findings.