Elevation Of Extracellular Potassium Concentration Research Articles

KCNQ2 channels regulate the population activity of neonatal GABAergic neurons ex vivo.

Over the last decade KCNQ2 channels have arisen as fundamental and indispensable regulators of neonatal brain excitability, with KCNQ2 loss-of-function pathogenic variants being increasingly identified in patients with developmental and epileptic encephalopathy. However, the mechanisms by which KCNQ2 loss-of-function variants lead to network dysfunction are not fully known. An important remaining knowledge gap is whether loss of KCNQ2 function alters GABAergic interneuron activity early in development. To address this question, we applied mesoscale calcium imaging ex vivo in postnatal day 4-7 mice lacking KCNQ2 channels in interneurons (Vgat-ires-cre;Kcnq2f/f;GCamp5). In the presence of elevated extracellular potassium concentrations, ablation of KCNQ2 channels from GABAergic cells increased the interneuron population activity in the hippocampal formation and regions of the neocortex. We found that this increased population activity depends on fast synaptic transmission, with excitatory transmission promoting the activity and GABAergic transmission curtailing it. Together, our data show that loss of function of KCNQ2 channels from interneurons increases the network excitability of the immature GABAergic circuits, revealing a new function of KCNQ2 channels in interneuron physiology in the developing brain.

Resolving the reversed rate effect of calcium channel blockers on human induced pluripotent stem cell-derived cardiomyocytes and the impact on in vitro cardiac safety evaluation

Calcium channel blockers (CCBs), such as diltiazem, nifedipine, and verapamil, cause tachycardia effects on several commercially available human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), when tested in culture media provided by suppliers, rather than bradycardia effects, as seen in vivo. We found that in test conditions where Na+ current of hiPSC-CMs was reduced to certain threshold by either specific Na+ channel blocker tetrodotoxin (TTX), or by voltage-dependent inactivation using elevated extracellular potassium concentrations, CCBs produced bradycardia effects on hiPSC-CMs. However, elevated extracellular potassium concentrations or the presence of TTX did not change other pharmacological responses of hiPSC-CMs, including CCBs' effects on contraction intensity and duration; beating rate change by calcium channel opener FPL64176, HCN blocker ivabradine, and β-adrenergic agonist isoproterenol; and action potential duration prolongation by hERG channel blocker dofetilide. We concluded that action potentials of hiPSC-CMs, with regards to the CCB phenotype, were Na+ current driven. When Na+ channel availability was reduced to a critical level, their action potentials became Ca2+ current driven, and their responses to CCBs correlated well to those seen in vivo. Importantly, the corrected bradycardia effect of calcium channel block with our defined conditions will provide more reliable results in cardiac safety readouts of test compounds that integrate multiple effects including calcium channel inhibition.

Computational Model for Cross-Depolarization in DRG Neurons via Satellite Glial Cells using [K]o: Role of Kir4.1 Channels and Extracellular Leakage.

Satellite glial cells (SGCs) are glial cells found in the peripheral nervous system where they tightly envelop the somata of the primary sensory neurons such as dorsal root ganglion (DRG) neurons and nodose ganglion (NG) neurons. The somata of these neurons are generally compactly packed in their respective ganglia (DRG and NG). SGCs covering a neuron behave as an insulator of electrical activity from neighbouring neurons within the ganglion. Several studies have however shown that the somata show "cross-depolarization" (CD). Origin of CDs has been hypothesized to be chemical in nature: either from neurotransmitter release from both SGCs and somata or from elevation of extracellular potassium concentration ([K]o) in the vicinity of somata. Here, we investigate the role of Kir4.1 channels on SGC and diffusion/clearance factor (β) of [K]o from the space between SGC and DRG neuron somata to the bulk extracellular space in ganglion. We show using two "Soma-SGC Units" interacting via gap junction that a combination of Kir4.1 and β could be responsible for CD between DRG neuron somata in pathological conditions.

FRET-based imaging of intracellular ATP in organotypic brain slices.

Active neurons require a substantial amount of adenosine triphosphate (ATP) to re-establish ion gradients degraded by ion flux across their plasma membranes. Despite this fact, neurons, in contrast to astrocytes, do not contain any significant stores of energy substrates. Recent work has provided evidence for a neuro-metabolic coupling between both cell types, in which increased glycolysis and lactate production in astrocytes support neuronal metabolism. Here, we established the cell type-specific expression of the Förster resonance energy transfer (FRET) based nanosensor ATeam1.03YEMK ("Ateam") for dynamic measurement of changes in intracellular ATP levels in organotypic brain tissue slices. To this end, adeno-associated viral vectors coding for Ateam, driven by either the synapsin- or glial fibrillary acidic protein (GFAP) promoter were employed for specific transduction of neurons or astrocytes, respectively. Chemical ischemia, induced by perfusion of tissue slices with metabolic inhibitors of cellular glycolysis and mitochondrial respiration, resulted in a rapid decrease in the cellular Ateam signal to a new, low level, indicating nominal depletion of intracellular ATP. Increasing the extracellular potassium concentration to 8mM, thereby mimicking the release of potassium from active neurons, did not alter ATP levels in neurons. It, however, caused in an increase in ATP levels in astrocytes, a result which was confirmed in acutely isolated tissue slices. In summary, our results demonstrate that organotypic cultured slices are a reliable tool for FRET-based dynamic imaging of ATP in neurons and astrocytes. They moreover provide evidence for an increased ATP synthesis in astrocytes, but not neurons, during periods of elevated extracellular potassium concentrations.

Resolving the Reversed Rate Effect of Calcium Channel Blockers on Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes and the Impact on In Vitro Cardiac Safety Evaluation.

Calcium channel blockers (CCBs), such as diltiazem, nifedipine, and verapamil, cause tachycardia effects on several commercially available human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), when tested in culture media provided by suppliers, rather than bradycardia effects, as seen in vivo. We found that in test conditions where Na+ current of hiPSC-CMs was reduced to certain threshold by either specific Na+ channel blocker tetrodotoxin (TTX), or by voltage-dependent inactivation using elevated extracellular potassium concentrations, CCBs produced bradycardia effects on hiPSC-CMs. However, elevated extracellular potassium concentrations or the presence of TTX did not change other pharmacological responses of hiPSC-CMs, including CCBs' effects on contraction intensity and duration; beating rate change by calcium channel opener FPL64176, HCN blocker ivabradine, and β-adrenergic agonist isoproterenol; and action potential duration prolongation by hERG channel blocker dofetilide. We concluded that action potentials of hiPSC-CMs, with regards to the CCB phenotype, were Na+ current driven. When Na+ channel availability was reduced to a critical level, their action potentials became Ca2+ current driven, and their responses to CCBs correlated well to those seen in vivo. Importantly, the corrected bradycardia effect of calcium channel block with our defined conditions will provide more reliable results in cardiac safety readouts of test compounds that integrate multiple effects including calcium channel inhibition.

Different patterns of epileptiform-like activity are generated in the sclerotic hippocampus from patients with drug-resistant temporal lobe epilepsy

Human hippocampal slice preparations from patients with temporal lobe epilepsy (TLE) associated with hippocampal sclerosis (HS) are excellent material for the characterization of epileptiform-like activity. However, it is still unknown if hippocampal regions as cornu Ammonis (CA) 1, CA3 and CA4, generate population epileptiform-like activity. Here, we investigated epileptiform activities of the subiculum, CA1, CA2, CA3, CA4 (induced by elevation of extracellular potassium concentration) and the dentate gyrus (induced with hilar stimulation and elevation of potassium concentration) from sclerotic hippocampi of patients with drug-resistant TLE. Five types of epileptiform-like activity were observed: interictal-like events; periodic ictal spiking; seizure-like events; spreading depression-like events; tonic seizure-like events and no activity. Different susceptibilities to generate epileptiform activity among hippocampal regions were observed; the dentate gyrus was the most susceptible region followed by the subiculum, CA4, CA1, CA2 and CA3. The incidence of epileptiform activity pattern was associated with specific regions of the hippocampal formation. Moreover, it was observed that each region of the hippocampal formation exhibits frequency-specific ranges in each subfield of the sclerotic human tissue. In conclusion, this study demonstrates that epileptiform-like activity may be induced in different regions of the hippocampal formation, including regions that are severely affected by neuronal loss.

Preferential Initiation and Spread of Anoxic Depolarization in Layer 4 of Rat Barrel Cortex.

Anoxic depolarization (AD) is a hallmark of ischemic brain damage. AD is associated with a spreading wave of neuronal depolarization and an increase in light transmittance. However, initiation and spread of AD across the layers of the somatosensory cortex, which is one of the most frequently affected brain regions in ischemic stroke, remains largely unknown. Here, we explored the initiation and propagation of AD in slices of the rat barrel cortex using extracellular local field potential (LFP) recordings and optical intrinsic signal (OIS) recordings. We found that ischemia-like conditions induced by oxygen-glucose deprivation (OGD) evoked AD, which manifested as a large negative LFP shift and an increase in light transmittance. AD typically initiated in one or more barrels and further spread across the entire slice with a preferential propagation through L4. Elevated extracellular potassium concentration accelerated the AD onset without affecting proneness of L4 to AD. In live slices, barrels were most heavily labeled by the metabolic level marker 2,3,5-triphenyltetrazolium chloride, suggesting that the highest metabolic demand is in L4 when compared to the other layers. Thus, L4 is the layer of the barrel cortex most prone to AD, which may be due to the highest metabolic demand and cell density in this layer.

Rotenone and elevated extracellular potassium concentration induce cell-specific fibrillation of α-synuclein in axons of cholinergic enteric neurons in the guinea-pig ileum.

Parkinson's disease is a progressive neurodegenerative disorder that results in the widespread loss of select classes of neurons throughout the nervous system. The pathological hallmarks of Parkinson's disease are Lewy bodies and neurites, of which α-synuclein fibrils are the major component. α-Synuclein aggregation has been reported in the gut of Parkinson's disease patients, even up to a decade before motor symptoms, and similar observations have been made in animal models of disease. However, unlike the central nervous system, the nature of α-synuclein species that form these aggregates and the classes of neurons affected in the gut are unclear. We have previously reported selective expression of α-synuclein in cholinergic neurons in the gut (J Comp Neurol. 2013; 521:657), suggesting they may be particularly vulnerable to degeneration in Parkinson's disease. In this study, we used immunohistochemistry to detect α-synuclein oligomers and fibrils via conformation-specific antibodies after rotenone treatment or prolonged exposure to high [K+ ] in ex vivo segments of guinea-pig ileum maintained in organotypic culture. Rotenone and prolonged raising of [K+ ] caused accumulation of α-synuclein fibrils in the axons of cholinergic enteric neurons. This took place in a time- and, in the case of rotenone, concentration-dependent manner. Rotenone also caused selective necrosis, indicated by increased cellular autofluorescence, of cholinergic enteric neurons, labeled by ChAT-immunoreactivity, also in a concentration-dependent manner. To our knowledge, this is the first report of rotenone causing selective loss of a neurochemical class in the enteric nervous system. Cholinergic enteric neurons may be particularly susceptible to Lewy pathology and degeneration in Parkinson's disease.

Management of hyperkalaemia

Hyperkalaemia, an elevated extracellular fluid potassium concentration, is a common electrolyte disorder and is present in 1-10% of hospitalised patients. Elevated serum potassium concentrations are usually asymptomatic but may be associated with electrocardiogram (ECG) changes. Hyperkalaemia occasionally leads to life-threatening cardiac arrhythmias. Prompt recognition of this disorder, patient risk management and administration of appropriate treatment can prevent serious cardiac complications of hyperkalaemia. Further assessment of the underlying basis for hyperkalaemia usually reveals a problem with renal potassium excretion (rather than transcellular shift of potassium or excess potassium intake). Reduced potassium excretion is typically associated with decreased potassium secretion in the aldosterone-sensitive distal nephron of the kidney. Common causes for hyperkalaemia include kidney failure, limited delivery of sodium and water to the distal nephron and drugs that inhibit the renin-angiotensin-aldosterone system. Treatment of life-threatening hyperkalaemia (particularly those patients with ECG changes) involves administration of intravenous calcium salts to stabilise the resting cardiac membrane potential. The potassium concentration can be lowered by administration of intravenous insulin combined with an infusion of glucose to stimulate intracellular uptake of potassium. Nebulised β-2 adrenoceptor agonists can augment the effects of intravenous insulin and glucose pending more definitive management of the recurrent hyperkalaemia risk. Additional management steps include stopping further potassium intake and careful review of prescribed drugs that may be adversely affecting potassium homeostasis. Changes to prescribing systems and an agreed institutional protocol for management of hyperkalaemia can improve patient safety for this frequently encountered electrolyte disorder.

Optogenetic Control of Vascular Tone with High Temporal Resolution

Blood pressure is directly controlled by the contractile state of vascular smooth muscle cells (VSMCs) which is regulated by membrane potential-dependent Ca2+-entry through Ca2+-channels and Ca2+-release from intracellular stores. Modulation of membrane potential in VSMCs in intact vessels can be performed by electrical field stimulation or elevation of extracellular potassium concentration, but both methods are not cell-specific and have low spatio-temporal resolution. To overcome these limitations, we have used the light-gated cation channel channelrhodopsin2 for optogenetical control of VSMCs membrane potential. ChR2 was expressed in a7r5 VSMCs and patch clamp experiments showed light-induced inward currents. Brief light pulses with increasing light intensities led to graded depolarization and eventually initiation of action potentials. Prolonged illumination resulted in constant depolarization and elevated intracellular Ca2+-concentrations. Optogenetic stimulation of VSMCs in intact vessels was performed using transgenic mice that express ChR2 (Nat Methods. 2010;7:897-900) in VSMCs but not endothelial cells. Isometric force was analyzed in aortic rings with a wire-myograph. Illumination with blue light (475nm, 2.7mW/mm2) reliably induced contractions with very fast on- (< 800ms) and off-kinetics (< 2.5s) and contraction could be maintained up to 10 min. The amplitude of contraction could be graded by variation of light intensity and reached a maximum comparable to stimulation with noradrenalin (10μM). Control vessels from wild-type mice or transgenic mice expressing EGFP did not show light-induced contractions. Taken together ChR2 enables light-induced contraction of VSMCs with high temporal resolution and represents a powerful technique to analyze membrane-potential dependent mechanisms in VSMCs. Optogenetic stimulation allows cell-specific and localized stimulation down to single cell level and therefore can be used in the future to investigate the interplay of endothelial cells and VSMCs as well as the electrical coupling between cells within intact vessels.

Activation of Voltage-Sensitive Phosphatases with Pharmacological and Optogenetic Methods

Voltage-sensitivephosphatases (VSPs) are proteins that dephosphorylate phosphoinositides (PIs) upon membrane voltage dependent activation. Particularly, Ci-VSP (Ciona intestinalis Voltage-Sensitive Phosphatase) is a 5-phosphatase of PI(4,5)P2 and PI(3,4,5)P3 and has so far been studied by electrophysiological means.Here we developed more broadly applicable methods of membrane depolarization and therefore phosphatase activation. First, depolarization was achieved by coexpression of the K+ channels TASK and KCNQ and elevation of extracellular potassium concentration. Second, we utilized the capsaicin-activated TRPV1 channel, and last the photosensitive Channel Rhodopsin, ChR2, in order to activate Ci-VSP. Cultured cell lines were transfected with the different channels, Ci-VSP and sensors for PIs, e.g. the tubby and the PLCδ1-PH domain, each tagged with a fluorescent protein. Confocal microscopy was used to visualize and quantify the translocation of the PI-sensors from the membrane to the cytoplasm after the dephosphorylation of PIs. All three methods resulted in successful activation of Ci-VSP, with TRPV1 and ChR2 mediated depolarization to give the most robust results. Specifically, the ChR2 activation provides a very rapid response without the need of solution exchanges, simplifying the experimental procedures. To conclude, we suggest a series of methods that allow the manipulation of PI levels as well as the study of VSPs in living cells without electrophysiological instrumentation.This work was supported by a grant of the University Medical Center Giessen and Marburg (UKGM 32/2011 MR) to CRH and by Deutsche Forschungsgemeinschaft (SFB593 TP A12) to DO.

A Specific Two-pore Domain Potassium Channel Blocker Defines the Structure of the TASK-1 Open Pore

Two-pore domain potassium (K2P) channels play a key role in setting the membrane potential of excitable cells. Despite their role as putative targets for drugs and general anesthetics, little is known about the structure and the drug binding site of K2P channels. We describe A1899 as a potent and highly selective blocker of the K2P channel TASK-1. As A1899 acts as an open-channel blocker and binds to residues forming the wall of the central cavity, the drug was used to further our understanding of the channel pore. Using alanine mutagenesis screens, we have identified residues in both pore loops, the M2 and M4 segments, and the halothane response element to form the drug binding site of TASK-1. Our experimental data were used to validate a K2P open-pore homology model of TASK-1, providing structural insights for future rational design of drugs targeting K2P channels.

Control of Programmed Cell Death by Distinct Electrical Activity Patterns

Electrical activity and sufficient supply with survival factors play a major role in the control of apoptosis in the developing cortex. Coherent high-frequency neuronal activity, which efficiently releases neurotrophins, is essential for the survival of immature neurons. We studied the influence of neuronal activity on apoptosis in the developing cortex. Dissociated cultures of the newborn mouse cerebral cortex were grown on multielectrode arrays to determine the activity patterns that promote neuronal survival. Cultures were transfected with a plasmid coding for a caspase-3-sensitive fluorescent protein allowing real-time analysis of caspase-3-dependent apoptosis in individual neurons. Elevated extracellular potassium concentrations (5 and 8 mM), application of 4-aminopyridine or the γ-aminobutyric acid-A receptor antagonist Gabazine induced a shift in the frequency distribution of activity toward high-frequency bursts. Under these conditions, a reduction or delay in caspase-3 activation and an overall increase in neuronal survival could be observed. This effect was dependent on the activity of phosphatidylinositol-3 kinase, as blockade of this enzyme abolished the survival-promoting effect of high extracellular potassium concentrations. Our data indicate that increased network activity can prevent apoptosis in developing cortical neurons.

Partial Disinhibition Is Required for Transition of Stimulus-Induced Sharp Wave–Ripple Complexes Into Recurrent Epileptiform Discharges in Rat Hippocampal Slices

Sharp wave-ripple complexes (SPW-Rs) in the intact rodent hippocampus are characterized by slow field potential transients superimposed by close to 200-Hz ripple oscillations. Similar events have been recorded in hippocampal slices where SPW-Rs occur spontaneously or can be induced by repeated application of high-frequency stimulation, a standard protocol for induction of long-lasting long-term potentiation. Such stimulation is reminiscent of protocols used to induce kindling epilepsy and ripple oscillations may be predictive of the epileptogenic zone in temporal lobe epilepsy. In the present study, we investigated the relation between recurrent epileptiform discharges (REDs) and SPW-Rs by studying effects of partial removal of inhibition. In particular, we compared the effects of nicotine, low-dose bicuculline methiodide (BMI), and elevated extracellular potassium concentration ([K(+)](o)) on induced SPW-Rs. We show that nicotine dose-dependently transformed SPW-Rs into REDs. This transition was associated with reduced inhibitory conductance in CA3 pyramidal cells. Similar results were obtained from slices where the GABAergic conductance was reduced by application of low concentrations of BMI (1-2 μM). In contrast, sharp waves were diminished by phenobarbital. Elevating [K(+)](o) from 3 to 8.5 mM did not transform SPW-Rs into REDs but significantly increased their incidence and amplitude. Under these conditions, the equilibrium potential for inhibition was shifted in depolarizing direction, whereas inhibitory conductance was significantly increased. Interestingly, the propensity of elevated [K(+)](o) to induce seizure-like events was reduced in slices where SPW-Rs had been induced. In conclusion, recruitment of inhibitory cells during SPW-Rs may serve as a mechanism by which hyperexcitation and eventually seizure generation might be prevented.

The role of calcium influx pathways in phospholipase D activation in bovine adrenal glomerulosa cells

The steroid hormone aldosterone maintains sodium homeostasis and is therefore important in the control of blood volume and pressure. Angiotensin II (AngII) and elevated extracellular potassium concentrations ([K(+)](e)), the prime physiologic regulators of aldosterone secretion from adrenal glomerulosa cells, activate phospholipase D (PLD) in these cells. The role of Ca(2+) in the activation by these agents is unknown, although nitrendipine, a voltage-dependent Ca(2+) channel antagonist, does not inhibit AngII-elicited PLD activation, despite the fact that this compound blocked elevated [K(+)](e)-stimulated PLD activity. PLD activation triggered by AngII was also unaffected by the T-type calcium channel inhibitor nickel. Nevertheless, Ca(2+) influx was required for AngII-induced PLD activation in both primary cultures of bovine adrenal glomerulosa cells and a glomerulosa cell model, the NCI H295R adrenocortical carcinoma cell line. The involvement of store-operated Ca(2+) (SOC) influx and Ca(2+) release-activated Ca(2+) (CRAC) influx pathways in PLD activation was investigated using thapsigargin, an endoplasmic reticulum Ca(2+) pump inhibitor that empties the store to induce SOC influx, and the SOC inhibitor YM-58483 (BTP2), as well as a CRAC inhibitor, tyrphostin A9. In bovine glomerulosa cells, tyrphostin A9 inhibited AngII-induced PLD activation without affecting elevated [K(+)](e)-stimulated enzyme activity. On the other hand, differences were observed between the bovine adrenal glomerulosa and H295R cells in the involvement of Ca(2+) influx pathways in PLD activation, with the involvement of the SOC pathway suggested in the H295R cells. In summary, our results indicate that Ca(2+) entry only through certain Ca(2+) influx pathways is linked to PLD activation.

Nitric Oxide Modulates Spreading Depolarization Threshold in the Human and Rodent Cortex

Recent clinical data have suggested that prolonged cortical spreading depolarizations (CSDs) contribute to the pathogenesis of delayed ischemic neurologic deficits after subarachnoid hemorrhage. Elevated extracellular potassium concentrations and lowered nitric oxide (NO) levels have been detected in experimental and clinical subarachnoid hemorrhage. We investigated whether a similar extracellular composition renders the brain more susceptible to CSDs. Electrophysiologic and blood flow changes were studied in vivo in rats. Intrinsic optical signals, alterations of NO level, and electrophysiologic changes were investigated in rodent and human brain slices. Elevation of subarachnoid extracellular potassium in rats in vivo triggered CSDs. Using NO-sensitive dyes, we found that CSDs induce NO synthesis in neurons and endothelial cells. When we blocked NO synthesis in vivo, CSDs occurred at a significantly lower threshold and propagated with a wave of ischemia. This increased susceptibility for CSDs by a low NO level was confirmed in rat and human neocortical slices and depended on P/Q-type calcium channels and N-methyl-D-aspartate receptors, but not on guanylate cyclase. Mice deficient in endothelial NO synthase, in contrast to mice deficient in neuronal NO synthase, had an inherently lower threshold. Basal NO production determined CSD threshold. The threshold effect depended predominantly on endothelial NO synthase. Reduced NO levels, as in patients with subarachnoid hemorrhage, may render the brain more susceptible to CSDs. Because CSDs have been linked to the pathogenesis of delayed ischemic neurologic deficits, raising its threshold by increasing NO availability may prove therapeutically beneficial in patients with subarachnoid hemorrhage.

REGULATION OF PULMONARY VASOCONSTRICTION BY AGONISTS AND CAVEOLAE

Sustained pulmonary vasoconstriction contributes to the elevated pulmonary vascular resistance observed in pulmonary arterial hypertension. A rise in cytosolic Ca2 + in pulmonary artery smooth muscle cells (PASMCs) is major trigger for pulmonary vasoconstriction. One family of drugs currently being pursued as a potential treatment for pulmonary hypertension are the statins, which act by depleting cholesterol and reducing the number of caveolae. This study aimed at investigating the role of caveolae, membrane receptors, and ion channels (that are potentially located in the caveolae) in agonist-mediated pulmonary vasoconstriction in order to gain a greater understanding of the signaling mechanisms involved in the regulation of pulmonary vascular tone. Chronic treatment of PASMCs with the cholesterol-depleting agent, methyl-β -cyclodextrin (Mβ CD), significantly reduced the number of cholesterol rich caveolae regions in the membrane. This disruption of cholesterol in caveolae significantly inhibited pharmacomechanical (induced by phenylephrine), but not electromechanical (induced by elevated extracellular potassium concentration), rat pulmonary artery contraction. These results indicate that receptors may functionally colocalize in caveolae in PASMCs and coordinate to regulate pulmonary vascular tone.

Rhythms of high-grade block in an ionic model of a strand of regionally ischemic ventricular muscle

Electrical alternans, a beat-to-beat alternation in the electrocardiogram or electrogram, is frequently seen during the first few minutes of acute myocardial ischemia, and is often immediately followed by malignant cardiac arrhythmias such as ventricular tachycardia and ventricular fibrillation. As ischemia progresses, higher-order periodic rhythms (e.g., period-4) can replace the period-2 alternans rhythm. This is also seen in modelling work on a two-dimensional (2-D) sheet of regionally ischemic ventricular muscle. In addition, in the experimental work, ventricular arrhythmias are overwhelmingly seen only after the higher-order rhythms arise. We investigate an ionic model of a strand of ischemic ventricular muscle, constructed as a 3-cm-long 1-D cable with a centrally located 1-cm-long segment exposed to an elevated extracellular potassium concentration ([K +] o). As [K +] o is raised in this “ischemic segment” to represent one major effect of ongoing ischemia, the sequence of rhythms {1:1→2:2 (alternans)→2:1} is seen. With further increase in [K +] o, one sees higher-order periodic 2 N: M rhythms {2:1→4:2→4:1→6:2→6:1→8:2→8:1}. In a 2 N: M cycle, only M of the 2 N action potentials generated at the proximal end of the cable successfully traverse the ischemic segment, with the remaining ones being blocked within the ischemic segment. Finally, there is a transition to complete block {8:1→2:0→1:0} (in an n:0 rhythm, all action potentials die out within the ischemic segment). Changing the length of the ischemic segment results in different rhythms and transitions being seen: e.g., when the ischemic segment is 2 cm long, the period-6 rhythms are not seen; when it is 0.5 cm long, there is a 3:1 rhythm interposed between the 2:1 and 1:0 rhythms. We discuss the relevance of our results to the experimental observations on the higher-order rhythms that presage reentrant ischemic ventricular arrhythmias.

Coexistence of tonic firing and bursting in cortical neurons

Sustained neuronal activity can be broadly classified as either tonic firing or bursting. These two major patterns of neuronal oscillations are state dependent and may coexist. The dynamics and intracellular mechanisms of transitions between tonic firing and bursting in cortical networks remain poorly understood. Here we describe a detailed two-compartment conductance-based cortical neuron model which exhibits bistability with hysteresis between tonic firing and bursting for elevated extracellular potassium concentration. The study explains the ionic and dynamical mechanisms of burst generation and reveals the conditions underlying coexistence of two different oscillatory modes as a function of neuronal excitability.

Carbamazepine-resistance in the epileptic dentate gyrus of human hippocampal slices

Overexpression of drug efflux pumps at the blood brain barrier (BBB) has been suggested to be one important factor contributing to drug resistance in epilepsy. This would imply that resected brain tissue of drug-resistant patients is drug-sensitive in absence of the BBB. Here we studied the effects of carbamazepine (CBZ) at therapeutically relevant concentration on epileptiform activity electrophysiologically recorded in acute hippocampal slices of patients with mesial temporal lobe epilepsy (MTLE; 28 patients, 49 slices) or extra-hippocampal tumours (tumour; 6 patients, 11 slices). Epileptiform activity was induced by hilar stimulation (0.067 Hz) during elevation of extracellular potassium concentration ([K(+)](o)) and remained self-sustained in presence of 10-12 mM [K(+)](o). Quantitative analysis of data revealed that epileptiform activity in tissue of tumour-patients was predominantly suppressed by CBZ, indicating that the 'epilepsy model' used is CBZ-sensitive. In contrast, epileptiform activity in tissue of drug-resistant MTLE patients was resistant to CBZ in 82% of patients, partially suppressed in 11% and completely suppressed in 7%. The effects of CBZ in tissue of MTLE patients did not depend on the type of activity, hippocampal pathology, excitability of the tissue, or equilibration time of the drug. Considering that CBZ has direct access to all compartments of the slice, our results suggest that CBZ-resistance mechanisms are located within the parenchyma of the dentate gyrus and contribute to drug resistance in the majority of MTLE patients. BBB-located drug-resistance mechanisms per se may play a minor role in this region, because CBZ-sensitivity was only observed in 7% of CBZ-resistant patients.