Hyperpolarization-activated cation (HCN) channels are believed to be involved in the generation of cardiac pacemaker depolarizations as well as in the control of neuronal excitability and plasticity. The contributions of the four individual HCN channel isoforms (HCN1±4) to these diverse functions are not known. Here we show that HCN2-de®cient mice exhibit spontaneous absence seizures. The thalamocortical relay neurons of these mice displayed a near complete loss of the HCN current, resulting in a pronounced hyperpolarizing shift of the resting membrane potential, an altered response to depolarizing inputs and an increased susceptibility for oscillations. HCN2-null mice also displayed cardiac sinus dysrhythmia, a reduction of the sinoatrial HCN current and a shift of the maximum diastolic potential to hyperpolarized values. Mice with cardiomyocytespeci®c deletion of HCN2 displayed the same dysrhythmia as mice lacking HCN2 globally, indicating that the dysrhythmia is indeed caused by sinoatrial dysfunction. Our results de®ne the physiological role of the HCN2 subunit as a major determinant of membrane resting potential that is required for regular cardiac and neuronal rhythmicity.

The thalamocortical network is characterized by rhythmic burst activity during natural sleep and tonic single-spike activity during wakefulness. The change between these two activity modes is partially governed by transmitters acting on leak K+ currents in the thalamus, although the nature of the constituting ion channels is not yet known. In the present study, the contribution of members of the two-pore domain K+ channel family to the leak current was investigated using whole-cell patch-clamp techniques and molecular biological techniques. RT-PCR and in situ hybridization revealed the expression of TWIK-related acid-sensitive K+ channel 1 (TASK 1) and TASK3 channels in the rat dLGN. Voltage-clamp recordings of thalamocortical relay neurons in slice preparations demonstrated the existence of a current component sensitive to the TASK channel blocker bupivacaine, which reversed at the presumed K+ equilibrium potential, showed outward rectification, and contributed approximately 40% to the standing outward current at depolarized values of the membrane potential (-28 mV). The pharmacological profile was indicative of TASK channels, in that the current was sensitive to changes in extracellular pH, reduced by muscarine and increased by halothane, and these effects were occluded by a near-maximal action of bupivacaine. Pharmacological manipulation of this current under current-clamp conditions resulted in a shift between burst and tonic firing modes. It is concluded that TASK1 and TASK3 channels contribute to the muscarine- and halothane-sensitive conductance in thalamocortical relay neurons, thereby contributing to the change in the activity mode of thalamocortical networks observed during the sleep-wake cycle and on application of inhalational anesthetics.

Functional neuroanatomy of Pavlovian fear has identified neuronal circuits and synapses associating conditioned stimuli with aversive events. Hebbian plasticity within these networks requires additional reinforcement to store particularly salient experiences into long-term memory. Here we have identified a circuit that reciprocally connects the ventral periaqueductal gray and dorsal raphe region with the central amygdala and that gates fear learning. We found that ventral periaqueductal gray and dorsal raphe dopaminergic (vPdRD) neurons encode a positive prediction error in response to unpredicted shocks and may reshape intra-amygdala connectivity via a dopamine-dependent form of long-term potentiation. Negative feedback from the central amygdala to vPdRD neurons might limit reinforcement to events that have not been predicted. These findings add a new module to the midbrain dopaminergic circuit architecture underlying associative reinforcement learning and identify vPdRD neurons as a critical component of Pavlovian fear conditioning. We propose that dysregulation of vPdRD neuronal activity may contribute to fear-related psychiatric disorders.

The role of hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channel isoforms and hyperpolarization-activated cation current (I h ) for seizure-related burst firing in thalamocortical (TC) neurons was investigated in a rat genetic model of absence epilepsy [Wistar Albino Glaxo rats, bred in Rijswijk (WAG/Rij)]. Burst discharges in TC neurons locked to seizure activity in vivo were prolonged during blockade of I h by Cs ϩ and ZD7288 (4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride). In vitro analyses revealed a hyperpolarizing shift of half-maximal I h activation (V h ) in WAG/Rij (V h ϭ Ϫ93.2 mV) compared with nonepileptic controls [August ϫ Copenhagen-Irish (ACI) (V h ϭ Ϫ88.0 mV)]. This effect is explained by a shift of the responsiveness of I h to cAMP toward higher concentrations in TC neurons from WAG/Rij, as revealed by application of 8-bromo-cAMP and the phosphodiesterase inhibitor IBMX. During blockade of adenylyl cyclase activity, I h activation was similar in the two strains, whereas the difference in cAMP responsiveness persisted, thereby voting against different ambient cAMP levels between strains. Increasing the intracellular cAMP level and shifting I h activation led to a change from burst to tonic firing mode in WAG/Rij but not in ACI rats. Furthermore, HCN1 expression was significantly increased on mRNA and protein levels, with no changes in HCN2-4 expression. In conclusion, there is an increase in HCN1 expression in the epileptic thalamus, associated with a decrease in cAMP responsiveness of I h in TC neurons and resulting impairment to control the shift from burst to tonic firing, which, in turn, will prolong burst activity after recruitment of I h during absence seizures.

By combining molecular biological, electrophysiological, immunological, and computer modeling techniques, we here demonstrate a counterbalancing contribution of TASK channels, underlying hyperpolarizing K+ leak currents, and HCN channels, underlying depolarizing Ih, to the resting membrane potential of thalamocortical relay (TC) neurons. RT-PCR experiments revealed the expression of TASK1, TASK3, and HCN1-4. Quantitative determination of mRNA expression levels and immunocytochemical staining demonstrated that TASK3 and HCN2 channels represent the dominant thalamic isoforms and are coexpressed in TC neurons. Extracellular acidification, a standard procedure to inhibit TASK channels, blocked a TASK current masked by additional action on HCN channels. Only in the presence of the HCN blocker ZD7288 was the pH-sensitive component typical for a TASK current, i.e., outward rectification and current reversal at the K+ equilibrium potential. In a similar way extracellular acidification was able to shift the activity pattern of TC neurons from burst to tonic firing only during block of Ih or genetic knock out of HCN channels. A single compartmental computer model of TC neurons simulated the counterbalancing influence of TASK and HCN on the resting membrane potential. It is concluded that TASK3 and HCN2 channels stabilize the membrane potential by a mutual functional interaction, that the most efficient way to regulate the membrane potential of TC neurons is the converse modulation of TASK and HCN channels, and that TC neurons are potentially more resistant to insults accompanied by extracellular pH shifts in comparison to other CNS regions.

Phosphatidylinositol 4-OH kinase III␤ (PI-4K␤) is involved in the regulated local synthesis of phospholipids that are crucial for trans-Golgi network (TGN)-to-plasma membrane trafficking. In this study, we show that the calcium sensor proteins calneuron-1 and calneuron-2 physically associate with PI-4K␤, inhibit the enzyme profoundly at resting and low calcium levels, and negatively interfere with Golgi-to-plasma membrane trafficking. At high calcium levels this inhibition is released and PI-4K␤ is activated via a preferential association with neuronal calcium sensor-1 (NCS-1). In accord to its supposed function as a filter for subthreshold Golgi calcium transients, neuronal overexpression of calneuron-1 enlarges the size of the TGN caused by a build-up of vesicle proteins and reduces the number of axonal Piccolo-Bassoon transport vesicles, large dense core vesicles that carry a set of essential proteins for the formation of the presynaptic active zone during development. A corresponding protein knockdown has the opposite effect. The opposing roles of calneurons and NCS-1 provide a molecular switch to decode local calcium transients at the Golgi and impose a calcium threshold for PI-4K␤ activity and vesicle trafficking.calcium binding protein 7 ͉ caldendrin ͉ neuronal calcium sensor-1 ͉ phosphatidylinositol 4-OH kinase III␤ ͉ calcium binding protein 8

Neuropeptide S (NPS) and its receptor are thought to define a set of specific brain circuits involved in fear and anxiety. Here we provide evidence for a novel, NPS-responsive circuit that shapes neural activity in the mouse basolateral amygdala (BLA) via the endopiriform nucleus (EPN). Using slice preparations, we demonstrate that NPS directly activates an inward current in 20% of EPN neurons and evokes an increase of glutamatergic excitation in this nucleus. Excitation of the EPN is responsible for a modulation of BLA activity through NPS, characterized by a general increase of GABAergic inhibition and enhancement of spike activity in a subset of BLA projection neurons. Finally, local injection of NPS to the EPN interferes with the expression of contextual, but not auditory cued fear memory. Together, these data suggest the existence of a specific NPS-responsive circuitry between EPN and BLA, likely involved in contextual aspects of fear memory.

Cytotoxic CD8ϩ T cells are considered important effector cells contributing to neuronal damage in inflammatory and degenerative CNS disorders. Using time-lapse video microscopy and two-photon imaging in combination with whole-cell patch-clamp recordings, we here show that major histocompatibility class I (MHC I)-restricted neuronal antigen presentation and T cell receptor specificity determine CD8 ϩ T-cell locomotion and neuronal damage in culture and hippocampal brain slices. Two separate functional consequences result from a direct cell-cell contact between antigen-presenting neurons and antigen-specific CD8 ϩ T cells. (1) An immediate impairment of electrical signaling in single neurons and neuronal networks occurs as a result of massive shunting of the membrane capacitance after insertion of channel-forming perforin (and probably activation of other transmembrane conductances), which is paralleled by an increase of intracellular Ca 2ϩ levels (within Ͻ10 min). (2) Antigen-dependent neuronal apoptosis may occur independently of perforin and members of the granzyme B cluster (within ϳ1 h), suggesting that extracellular effects can substitute for intracellular delivery of granzymes by perforin. Thus, electrical silencing is an immediate consequence of MHC I-restricted interaction of CD8 ϩ T cells with neurons. This mechanism is clearly perforin-dependent and precedes, but is not causally linked, to neuronal cell death.