Biomedical subjects

Richard Kovács

Publications and source records attributed to Richard Kovács.

7 recordsLinked to original sources

PubMed2006

Mitochondria and neuronal activity.

Mitochondria are central for various cellular processes that include ATP production, intracellular Ca(2+) signaling, and generation of reactive oxygen species. Neurons critically depend on mitochondrial function to establish membrane excitability and to execute the complex processes of neurotransmission and plasticity. While much information about mitochondrial properties is available from studies on isolated mitochondria and dissociated cell cultures, less is known about mitochondrial function in intact neurons in brain tissue. However, a detailed description of the interactions between mitochondrial function, energy metabolism, and neuronal activity is crucial for the understanding of the complex physiological behavior of neurons, as well as the pathophysiology of various neurological diseases. The combination of new fluorescence imaging techniques, electrophysiology, and brain slice preparations provides a powerful tool to study mitochondrial function during neuronal activity, with high spatiotemporal resolution. This review summarizes recent findings on mitochondrial Ca(2+) transport, mitochondrial membrane potential (DeltaPsi(m)), and energy metabolism during neuronal activity. We will first discuss interactions of these parameters for experimental stimulation conditions that can be related to the physiological range. We will then describe how mitochondrial and metabolic dysfunction develops during pathological neuronal activity, focusing on temporal lobe epilepsy and its experimental models. The aim is to illustrate that 1) the structure of the mitochondrial compartment is highly dynamic in neurons, 2) there is a fine-tuned coupling between neuronal activity and mitochondrial function, and 3) mitochondria are of central importance for the complex behavior of neurons.

Animals↗

PubMed2006

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.

Action Potentials↗

PubMed2005

Metabolic dysfunction during neuronal activation in the ex vivo hippocampus from chronic epileptic rats and humans.

Metabolic dysfunction has been implicated in the pathogenesis of temporal lobe epilepsy (TLE), but its manifestation during neuronal activation in the ex vivo hippocampus from TLE patients has not been shown. We characterized metabolic and mitochondrial functions in acute hippocampal slices from pilocarpine-treated, chronic epileptic rats and from pharmaco-resistant TLE patients. Recordings of NAD(P)H fluorescence indicated the status of cellular energy metabolism, and simultaneous monitoring of extracellular potassium concentration ([K+]o) allowed us to control the induction of neuronal activation. In control rats, electrical stimulation elicited biphasic NAD(P)H fluorescence transients that were characterized by a brief initial 'drop' and a subsequent prolonged 'overshoot' correlating to enhanced NAD(P)+ reduction. In chronic epileptic rats, overshoots were significantly smaller in area CA1, but not in the subiculum as compared to controls. In TLE patients, who were histopathologically classified in groups with and without Ammon's horn sclerosis (AHS, non-AHS), large drops and very small overshoots of NAD(P)H transients were observed in dentate gyrus, CA3, CA1 and subiculum. Nevertheless, monitoring mitochondrial membrane potential (DeltaPsi(m)) by mitochondria-specific, voltage-sensitive dye (rhodamine-123) revealed similar mitochondrial responses during neuronal activation with glutamate and protonophore application in area CA1 of control and chronic-epileptic rats. Applying confocal laser scanning microscopy, these findings were confirmed in individual neurons of AHS tissue, indicating a negative DeltaPsi(m) and activation-dependent mitochondrial depolarization. Our data demonstrate severe metabolic dysfunction during neuronal activation in the hippocampus from chronic epileptic rats and humans, although mitochondria maintain negative DeltaPsi(m). Thus, our findings provide a cellular correlate for 'hypometabolism' as described for epilepsy patients and suggest mitochondrial enzyme defects in TLE.

Adult↗

PubMed2005

Mitochondrial calcium ion and membrane potential transients follow the pattern of epileptiform discharges in hippocampal slice cultures.

Emerging evidence suggests that mitochondrial dysfunction contributes to the pathophysiology of epilepsy. Recurrent mitochondrial Ca2+ ion load during seizures might act on mitochondrial membrane potential (DeltaPsim) and proton motive force. By using electrophysiology and confocal laser-scanning microscopy, we investigated the effects of epileptiform activity, as induced by low-Mg2+ ion perfusion in hippocampal slice cultures, on changes in DeltaPsim and in mitochondrial Ca2+ ion concentration ([Ca2+]m). The mitochondrial compartment was identified by monitoring DeltaPsim in the soma and dendrites of patched CA3 pyramidal cells using the mitochondria-specific voltage-sensitive dye rhodamine-123 (Rh-123). Interictal activity was accompanied by localized mitochondrial depolarization that was restricted to a few mitochondria in small dendrites. In contrast, robust Rh-123 release into the cytosol was observed during seizure-like events (SLEs), indicating simultaneous depolarization of mitochondria. This was critically dependent on Ca2+ ion uptake and extrusion, because inhibition of the mitochondrial Ca2+ ion uniporter by Ru360 and the mitochondrial Na+/Ca2+ ion exchanger by 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one but not the inhibitor of mitochondrial permeability transition pore, cyclosporin A, decreased the SLE-associated mitochondrial depolarization. The Ca2+ ion dependence of simultaneous mitochondrial depolarization suggested enhanced Ca2+ ion cycling across mitochondrial membranes during epileptiform activity. Indeed, [Ca2+]m fluctuated during interictal activity in single dendrites, and these fluctuations spread over the entire mitochondrial compartment during SLEs, as revealed using mitochondria-specific dyes (rhod-2 and rhod-ff) and spatial frequency-based image analysis. These findings strengthen the hypothesis that epileptic activity results in Ca2+ ion-dependent changes in mitochondrial function that might contribute to the neuronal injury during epilepsy.

Animals↗

PubMed2004

Hyperoxia is not an essential condition for status epilepticus induced cell death in organotypic hippocampal slice cultures.

The low Mg2+ model of epilepsy in organotypic hippocampal slice cultures is used to elucidate the mechanism underlying neuronal cell death following sustained epileptiform activity. However, the high oxygen tension of 95% widely used in this model is capable of inducing neuronal cell death by itself. Here we demonstrate that even under normoxic conditions 1h of epileptiform activity induced neuronal cell death as assessed by Propidium Iodide uptake. We conclude that hyperoxia is not essential for status epilepticus induced neuronal cell death in this model.

Animals↗

PubMed2003

Metabotropic receptor-mediated Ca2+ signaling elevates mitochondrial Ca2+ and stimulates oxidative metabolism in hippocampal slice cultures.

Metabotropic receptors modulate numerous cellular processes by intracellular Ca2+ signaling, but less is known about their role in regulating mitochondrial metabolic function within the CNS. In this study, we demonstrate in area CA3 of rat organotypic hippocampal slice cultures that glutamatergic, serotonergic, and muscarinic metabotropic receptor ligands, namely trans-azetidine-2,4-dicarboxylic acid, alpha-methyl-5-hydroxytryptamine, and carbachol, transiently increase mitochondrial Ca2+ concentration ([Ca2+]m) as recorded by changes in Rhod-2 fluorescence, stimulate mitochondrial oxidative metabolism as revealed by elevations in NAD(P)H fluorescence, and induce K+ outward currents as monitored by rapid increases in extracellular K+ concentration ([K+]o). Carbachol (1-1,000 microM) elevated NAD(P)H fluorescence by <or=14%DeltaF/F0 and increased [K+]o by <or=4.3 mM in a dose-dependent manner. Carbachol-induced responses persisted in Ca2+-free solution and blockade of ionotropic glutamatergic and nicotinic receptors. Under similar conditions caffeine, known to cause Ca2+-induced Ca2+ release (CICR), also evoked elevations in [Ca2+]m, NAD(P)H fluorescence and [K+]o that, in contrast to carbachol-induced responses, displayed oscillations. After depletion of intracellular Ca2+ stores by carbachol in Ca2+-free solution, re-application of 1.6 mM Ca2+-containing solution triggered marked elevations in [Ca2+]m, NAD(P)H fluorescence and [K+]o. These data indicate that metabotropic transmission effectively regulates mitochondrial oxidative metabolism via diverse receptor types in hippocampal cells and that inonitol 1,4,5-trisphosphate-induced Ca2+ release (IICR) or CICR or capacitative Ca2+ entry might suffice in stimulating oxidative metabolism by elevating [Ca2+]m. Thus activation of metabotropic receptors might significantly contribute to generation of ATP within neurons and glial cells.

Animals↗

PubMed2002

Free radical-mediated cell damage after experimental status epilepticus in hippocampal slice cultures.

Generation of free radicals may have a key role in the nerve cell damage induced by prolonged or frequently recurring convulsions (status epilepticus). Mitochondrial function may also be altered due to production of free radicals during seizures. We therefore studied changes in field potentials (fp) together with measurements of extracellular, intracellular, and intramitochondrial calcium concentration ([Ca(2+)]e, [Ca(2+)]i, and [Ca(2+)]m, respectively), mitochondrial membrane potential (deltapsi), NAD(P)H auto-fluorescence, and dihydroethidium (HEt) fluorescence in hippocampal slice cultures by means of simultaneous electrophysiological and microfluorimetric measurements. As reported previously, each seizure-like event (SLE) resulted in mitochondrial depolarization associated with a delayed rise in oxidation of HEt to ethidum, presumably indicating ROS production. We show here that repeated SLEs led to a decline in intracellular and intramitochondrial Ca(2+) signals despite unaltered Ca(2+) influx. Also, mitochondrial depolarization and the NAD(P)H signal became smaller during recurring SLEs. By contrast, the ethidium fluorescence rises remained constant or even increased from SLE to SLE. After about 15 SLEs, activity changed to continuous afterdischarges with steady depolarization of mitochondrial membranes. Staining with a cell death marker, propidium iodide, indicated widespread cell damage after 2 h of recurring SLEs. The free radical scavenger, alpha-tocopherol, protected the slice cultures against this damage and also reduced the ongoing impairment of NAD(P)H production. These findings suggest involvement of reactive oxygen species (ROS) of mitochondrial origin in the epileptic cell damage and that free radical scavenging may prevent status epilepticus-induced cell loss.

Animals↗