Thomas Munsch

Absence epilepsy and sinus dysrhythmia in mice lacking the pacemaker channel HCN2

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‐deficient 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 cardiomyocyte‐ specific deletion of HCN2 displayed the same dysrhythmia as mice lacking HCN2 globally, indicating that the dysrhythmia is indeed caused by sinoatrial dysfunction. Our results define the physiological role of the HCN2 subunit as a major determinant of membrane resting potential that is required for regular cardiac and neuronal rhythmicity.

Identification of a Neuropeptide S Responsive Circuitry Shaping Amygdala Activity via the Endopiriform Nucleus

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.

Impaired Regulation of Thalamic Pacemaker Channels through an Imbalance of Subunit Expression in Absence Epilepsy

The role of hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channel isoforms and hyperpolarization-activated cation current (Ih) 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 Ih by Cs+ and ZD7288 (4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride). In vitro analyses revealed a hyperpolarizing shift of half-maximal Ih activation (Vh) in WAG/Rij (Vh = -93.2 mV) compared with nonepileptic controls [August × Copenhagen-Irish (ACI) (Vh = -88.0 mV)]. This effect is explained by a shift of the responsiveness of Ih 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, Ih 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 Ih 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 Ih 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 Ih during absence seizures.

Contribution of transient receptor potential channels to the control of GABA release from dendrites

Neuronal dendrites have been shown to actively contribute to synaptic information transfer through the Ca2+-dependent release of neurotransmitter, although the underlying mechanisms remain elusive. This study shows that the increase in dendritic γ-aminobutyric acid (GABA) release from thalamic interneurons mediated by the activation of 5-hydroxytryptamine type 2 receptors requires Ca2+ entry that does not involve Ca2+ release nor voltage-gated Ca2+ channels in the plasma membrane but that is critically dependent on the transient receptor potential (TRP) protein TRPC4. These data ascribe a functional role of agonist-activated TRP channels to the release of transmitters from dendrites, thereby indicating a principle underlying synaptic interactions in the brain.

MODULATION OF THE HYPERPOLARIZATION-ACTIVATED CATION CURRENT OF RAT THALAMIC RELAY NEURONES BY INTRACELLULAR PH

1 Properties of the hyperpolarization‐activated cation current (Ih) were investigated in thalamocortical neurones of an in vitro slice preparation of the rat ventrobasal thalamic complex (VB) before and during changes of pipette pH (pHp), intracellular pH (pHi) and bath pH (pHb) using the whole‐cell patch‐clamp technique and fluorescence ratio imaging of the pH indicator 2′,7′‐bis(carboxyethyl)‐5(and ‐6)‐carboxyfluorescein (BCECF). 2 Recording of Ih with predefined pHp revealed significant shifts in the voltage dependence of Ih activation (V½) of 4‐5 mV to more positive values for a pHp of 7.5 and 2‐3 mV to more negative values for a pHp of 6.7 as compared to control values (pHp= 7.1). 3 Application of the weak acid lactate (20 mM), which produced a slow monophasic intracellular acidification, induced a reversible negative shift of V½ of up to 3 mV. Application of 20 mM TMA, which caused a distinct intracellular alkalinization, shifted V½ to 4‐5 mV more positive values. 4 In slices bathed in Hepes‐buffered saline, no significant pHo dependence of Ih was observed. Changing pHo by altering the extracellular [HCO3−] in the presence of constant pCO2 also revealed no significant pHo dependence of Ih. 5 Rhythmic stimulation of thalamocortical neurones with repetitive depolarizing pulse trains caused an intracellular acidification, which reversibly decreased the amplitude and time course of activation of Ih. 6 The results of the present study indicate that shifts in pHi result in a significant modulation of the gating properties of Ih channels in TC neurones. Through this mechanism activity‐dependent shifts in pHi may contribute to the up‐ and downregulation of Ih.

Voltage-activated intracellular calcium transients in thalamic relay cells and interneurons.

THE dynamics of intracellular calcium concentration ([Ca2+]i) following activation of low voltage-activated (LVA) and high voltage-activated (HVA) Ca2+ currents were studied in identified relay neurons and interneurons of the rat dorsal lateral geniculate nucleus (LGNd) in situ using Ca2+ imaging and patch-clamp techniques. In relay neurons, [Ca2+]i transients associated with the LVA Ca2+ current showed a fairly homogeneous somatodendritic distribution, whereas HVA transients significantly decreased to 65% of the somatic value at 60 μm dendritic distance. In interneurons, LVA transients significantly increased to 239% of the somatic value at 60 μm dendritic distance, whereas HVA transients were not significantly different in the soma and dendrites. These results indicate differences in [Ca2+]i dynamics, which may reflect a heterogeneous distribution of Ca2+ channels contributing to subcellular compartmentation in the two types of thalamic neurons.

Novel vistas of calcium-mediated signalling in the thalamus.

Traditionally, the role of calcium ions (Ca2+) in thalamic neurons has been viewed as that of electrical charge carriers. Recent experimental findings in thalamic cells have only begun to unravel a highly complex Ca2+ signalling network that exploits extra- and intracellular Ca2+ sources. In thalamocortical relay neurons, interactions between T-type Ca2+ channel activation, Ca2+-dependent regulation of adenylyl cyclase activity and the hyperpolarization-activated cation current (Ih) regulate oscillatory burst firing during periods of sleep and generalized epilepsy, while a functional triad between Ca2+ influx through high-voltage-activated (most likely L-type) Ca2+ channels, Ca2+-induced Ca2+ release via ryanodine receptors (RyRs) and a repolarizing mechanism (possibly via K+ channels of the BKCa type) supports tonic spike firing as required during wakefulness. The mechanisms seem to be located mostly at dendritic and somatic sites, respectively. One functional compartment involving local GABAergic interneurons in certain thalamic relay nuclei is the glomerulus, in which the dendritic release of GABA is regulated by Ca2+ influx via canonical transient receptor potential channels (TRPC), thereby presumably enabling transmitters of extrathalamic input systems that are coupled to phospholipase C (PLC)-activating receptors to control feed-forward inhibition in the thalamus. Functional interplay between T-type Ca2+ channels in dendrites and the A-type K+ current controls burst firing, contributing to the range of oscillatory activity observed in these interneurons. GABAergic neurons in the reticular thalamic (RT) nucleus recruit a specific set of Ca2+-dependent mechanisms for the generation of rhythmic burst firing, of which a particular T-type Ca2+ channel in the dendritic membrane, the Ca2+-dependent activation of non-specific cation channels (ICAN) and of K+ channels (SKCa type) are key players. Glial Ca2+ signalling in the thalamus appears to be a basic mechanism of the dynamic and integrated exchange of information between glial cells and neurons. The conclusion from these observations is that a localized calcium signalling network exists in all neuronal and probably also glial cell types in the thalamus and that this network is dedicated to the precise regulation of the functional mode of the thalamus during various behavioural states.

Lack of Regulation by Intracellular Ca2+ of the Hyper Polarization-Activated Cation Current in Rat Thalamic Neurones

1 The regulation of the hyperpolarization‐activated cation current, Ih, in thalamocortical neurones by intracellular calcium ions has been implemented in a number of mathematical models on the waxing and waning behaviour of synchronized rhythmic activity in thalamocortical circuits. In the present study, the Ca2+ dependence of Ih in thalamocortical neurones was experimentally investigated by combining Ca2+ imaging and patch‐clamp techniques in the ventrobasal thalamic complex (VB) in vitro. 2 Properties of Ih were analysed before and during rhythmic stimulation of Ca2+ entry by trains of depolarizing voltage pulses. Despite a significant increase in intracellular Ca2+ concentration ([Ca2+]i) from resting levels of 74 ± 23 nM to 251 ± 78 nM upon rhythmic stimulation, significant differences in the voltage dependence of Ih activation did not occur (half‐maximal activation at −86.4 ± l.3 mV vs.−85.2 ± 2.9 mV; slope of the activation curve, 11.2 ± 2.4 mV vs. 12.5 ± 2.5 mV). Recording of Ih with predefined values of [Ca2+]i (13.2 nM or 10.01 μm in the patch pipette) revealed no significant differences in the activation curve or the fully activated I–V relationship of Ih. 3 In comparison, stimulation of the intracellular cyclic adenosine monophosphate (cAMP) pathway induced a significantly positive shift in Ih voltage dependence of +5.1 ± l.9 mV, with no alteration in the fully activated I–V relationship. 4 These data argue against a direct regulation of Ih by intracellular Ca2+, and particularly do not support a primary role of Ca2+‐dependent modulation of the Ih channels in the waxing and waning of sleep spindle oscillations in thalamocortical neurones.

Cytotoxic CD8+ T Cell–Neuron Interactions: Perforin-Dependent Electrical Silencing Precedes But Is Not Causally Linked to Neuronal Cell Death

Cytotoxic CD8+ 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+ 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+ 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 Ca2+ levels (within <10 min). (2) Antigen-dependent neuronal apoptosis may occur independently of perforin and members of the granzyme B cluster (within ∼1 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+ T cells with neurons. This mechanism is clearly perforin-dependent and precedes, but is not causally linked, to neuronal cell death.

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.