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Biomedical subjects

Rüdiger Köhling

Publications and source records attributed to Rüdiger Köhling.

At least 19 recordsLinked to original sources
PubMed2006

Methodological approaches to exploring epileptic disorders in the human brain in vitro.

Brain surgery, and in particular epilepsy surgery, offers the unique opportunity to study viable human central nervous tissue in vitro. This does not only open a window to address the basic mechanisms underlying human disease, such as epilepsy, but it allows to venture into investigating neurophysiological functions per se. In the present paper, we describe the most commonly used methods in the electrophysiological (and, at least to some extent, also histochemical and molecular) analysis of human tissue in vitro. In addition, we consider the pitfalls and limitations of such studies, in particular regarding the issue of tissue sampling procedures and control experiments.

Biopsy↗
PubMed2006

The impact of astrocytic gap junctional coupling on potassium buffering in the hippocampus.

Astrocytic gap junctions have been suggested to contribute to spatial buffering of potassium in the brain. Direct evidence has been difficult to gather because of the lack of astrocyte-specific gap junction blockers. We obtained mice with coupling-deficient astrocytes by crossing conditional connexin43-deficient mice with connexin30(-/-) mice. Similar to wild-type astrocytes, genetically uncoupled hippocampal astrocytes displayed negative resting membrane potentials, time- and voltage-independent whole-cell currents, and typical astrocyte morphologies. Astrocyte densities were also unchanged. Using potassium-selective microelectrodes, we assessed changes in potassium buffering in hippocampal slices of mice with coupling-deficient astrocytes. We demonstrate that astrocytic gap junctions accelerate potassium clearance, limit potassium accumulation during synchronized neuronal firing, and aid in radial potassium relocation in the stratum lacunosum moleculare. Furthermore, slices of mice with coupling-deficient astrocytes displayed a reduced threshold for the generation of epileptiform events. However, it was evident that radial relocation of potassium in the stratum radiatum was not dependent on gap junctional coupling. We suggest that the perpendicular array of individual astrocytes in the stratum radiatum makes these cells ideally suited for spatial buffering of potassium released by pyramidal cells, independent of gap junctions. In general, a surprisingly large capacity for K+ clearance was conserved in mice with coupling-deficient astrocytes, indicating that gap junction-dependent processes only partially account for K+ buffering in the hippocampus.

Animals↗
PubMed2006

Electrophysiology in ischemic neocortical brain slices: species differences vs. influences of anaesthesia and preparation.

Ischemia models are indispensable for the evaluation of measures to be clinically applied to brain trauma or stroke patients. Slice models provide good control over experimental parameters and allow for comparative examinations of human and animal brain tissue. Experimental tissue, however, may be altered by anaesthesia, preparatory technique, and, in the case of human tissue, by underlying diseases. These influences on tissue behaviour under ischemia were examined electrophysiologically. Native rat tissue slices were prepared either immediately after decapitation (n = 13), during short ether/barbiturate narcosis (n = 18), or after two hours of inhalation anaesthesia (n = 12) imitating clinical narcosis. Tissue from rats in which generalized amygdala-kindled seizures had been triggered by electric stimulation (n = 10) was prepared according to the decapitation protocol, while human tissue (n = 10) was obtained during epilepsy or tumour surgery. Electrophysiological data (latency and amplitude of anoxic depolarization, recovery of evoked potentials) were recorded during ischemia simulation. Neither details of preparation or anaesthesia nor a history of epileptic fits were associated with significant changes of electrophysiological reactions under ischemia. Human tissue showed a significantly higher ability to uphold transmembrane ion gradients under ischemia. The ability of brain tissue to withstand ischemia is obviously species dependent. For the transfer of experimental results into clinical use it is important that interspecies differences alone can bring about a significant change of tissue behaviour.

Anesthesia↗
PubMed2006

Nidogen and nidogen-associated basement membrane proteins and neuronal plasticity.

Extracellular matrix (ECM) proteins are thought to subserve structural functions as, for example, tissue barriers as well as guidance structures during cell growth, differentiation and tissue repair. Deletion of basement membrane (BM) components results in malformations of different organs, including the brain. Recent data, however, suggest that interference with cellular membrane-associated proteins interacting with ECM can alter neuronal excitability and synaptic plasticity without obvious underlying structural damage. This does not only apply to classical ECM proteins such as laminin, reelin and tenascin, but also to molecules of a rather specialized ECM, the BM. Here, nidogen (also termed entactin) appears to subserve a function in neuronal plasticity. Nidogen ablation leads to epileptic activity in vivo and the appearance of spontaneous epileptiform activity in vitro. This raises the intriguing question whether the BM protein nidogen may directly influence neuronal function in the CNS, opening the possibility of modulatory mechanisms of synaptic plasticity and excitability reaching beyond classical processes confined to cellular interactions.

Animals↗
PubMed2005

Epileptiform activity preferentially arises outside tumor invasion zone in glioma xenotransplants.

Seizures occur commonly with brain tumors. The underlying mechanisms are not understood. We analyzed network and cellular excitability changes in tumor-invaded and sham neocortical tissue in vitro using a rat glioblastoma model. Rat C6 glioma cells were transplanted into rat neocortex, yielding diffusely invading gliomas resembling human glioblastomas. We hypothesized that network excitability would increase in regions neighboring the tumor, and that initiation of epileptic discharges might be correlated to a higher density of intrinsically bursting neurones. Voltage-sensitive dye imaging revealed epileptic activity to be initiated in paratumoral zones (1-2 mm from main tumor mass), in contrast to control tissue, where epileptic foci appeared randomly throughout the neocortex. Neuronal firing patterns revealed significantly more intrinsically bursting neurones within these initiation zones than in regions directly adjacent to the tumor or in control tissue. We conclude that gliomas are associated with a higher density of intrinsically bursting neurones, and that these may preferentially initiate epileptiform events.

Action Potentials↗
PubMed2005

Intrinsic excitability, synaptic potentials, and short-term plasticity in human epileptic neocortex.

Although studies of epileptic human hippocampus suggest changes of synaptic and intrinsic excitability, few changes, save the appearance of spontaneous field/synaptic potentials, are known in epileptic neocortical tissue. However, invasive EEG and histological studies suggest that neocortical tissue, even in mesial temporal lobe epilepsy, can play an important role as an irritative zone or extrahippocampal focus. We hypothesized that intrinsic neuronal and synaptic excitability, as well as short-term plasticity, are altered in neocortical areas, particularly with elevated K+ levels as occur during seizures. We analyzed neuronal firing properties, synaptic responses, and paired-pulse plasticity in human neocortical slices from tissue resected during epilepsy surgery, both under normal and under pathological conditions, i.e., after elevating K+ (4/8 mM), with rat neocortical slices as controls. Neuronal firing properties were not different. We did find, however, alterations of synaptic responsiveness in epileptic tissue, i.e., an elevated network excitability with K+ elevations, and reduction of paired-pulse depression.

Adult↗
PubMed2005

Optical imaging of epileptiform activity in experimentally induced cortical malformations.

Electrophysiological studies of human cortical dysplasia and rodent models revealed widespread hyperexcitability in the malformation itself as well as in its vicinity. We here analyzed the initiation of paroxysmal epileptiform activity using optical imaging of neuronal activity in rats with cortical malformations induced by neonatal freeze lesions. Brain slice preparations were incubated with the voltage-sensitive dye RH795 and neuronal activity was monitored using a fast-imaging photodiode array combined with standard field potential recordings. Spontaneous paroxysmal epileptiform activity emerged in all slices from animals with cortical malformations and sham-operated controls 20-40 min after omission of extracellular Mg(2+). Following electrophysiological and optical recordings, slices were histochemically processed. Using this approach, the present study demonstrated that in animals with freeze-lesion-induced focal cortical malformations, paroxysmal epileptiform activity always emerged from the dysplastic cortex and then spread to adjacent areas through superficial layers. This distribution of initiation sites was significantly different to sham-operated controls in which epileptogenic foci were located in various cytoarchitectonic areas. The present study indicates that following global changes in excitability, the dysplastic cortex itself is the main initiation site of paroxysmal epileptiform activity in animals with focal cortical malformations.

Animals↗
PubMed2005

Cellular and molecular mechanisms of epilepsy in the human brain.

Animal models have provided invaluable data for identifying the pathogenesis of epileptic disorders. Clearly, the relevance of these experimental findings would be strengthened by the demonstration that similar fundamental mechanisms are at work in the human epileptic brain. Epilepsy surgery has indeed opened the possibility to directly study the functional properties of human brain tissue in vitro, and to analyze the mechanisms underlying seizures and epileptogenesis. Here, we summarize the findings obtained over the last 40 years from electrophysiological, histochemical and molecular experiments made with the human brain tissue. In particular, this review will focus on (i) the synaptic and non-synaptic properties of neocortical neurons along with their ability to produce synchronous activity; (ii) the anatomical and functional alterations that characterize limbic structures in patients presenting with mesial temporal lobe epilepsy; (iii) the issue of antiepileptic drug action and resistance; and (iv) the pathophysiology of seizure genesis in Taylor's type focal cortical dysplasia. Finally, we will address some of the problems that are inherent to this type of experimental approach, in particular the lack of proper controls and possible strategies to obviate this limitation.

Animals↗
PubMed2005

Detection of electrophysiological indicators of neurotoxicity in human and rat brain slices by a three-dimensional microelectrode array.

Electrophysiological techniques for the assessment of in vitro neurotoxicology have several advantages over other currently-used methods (for example, morphological techniques), including the ability to detect damage at a very early stage. Novel recording techniques based on microelectrode arrays are available, and could improve recording power. In this study, we investigated how a three-dimensional microelectrode array detects the electrophysiological endpoints of neurotoxicity. We conclude that electrophysiology sensitively reveals neurotoxic actions, and that three-dimensional microelectrode arrays could be proposed for use in neurotoxicology as recording tools that allow easy and sensitive multisite recording, from both rodent and human brain tissue.

Animals↗
PubMed2004

Phase-locking characteristics of limbic P3 responses in hippocampal sclerosis.

Amplitudes of the P3 recorded invasively from the medial temporal lobe (MTL-P3) have been reported to be reduced on the side of a mediotemporal epileptogenic focus. This reduction has been attributed to the massive cell loss within the hippocampus associated with hippocampal sclerosis. It has remained unclear how functional connectivity between the hippocampus and rhinal cortex, as well as within the hippocampus, is altered in hippocampal sclerosis. To investigate this issue, we analyzed to what extent stimulus-related phase-locking and power changes within the low-frequency range (2-30 Hz) and within the gamma band (32-48 Hz), as well as rhinal-hippocampal phase synchronization contribute to the averaged MTL-P3 potentials. Event-related responses were recorded via bilateral depth electrodes in epilepsy patients with unilateral hippocampal sclerosis, who performed a visual oddball experiment. On the contralateral (nonsclerotic) side, successful target detection was associated with an increase of power and phase locking of hippocampal activity in both the low-frequency range and in the gamma range. Besides, there were rhinal-hippocampal synchronization enhancements in the theta and gamma range. On the ipsilateral (sclerotic) side, the event-related power increase in the low-frequency range had almost disappeared, a finding likely to be explained by the loss of principle neurons. However, low-frequency phase-locking, rhinal-hippocampal synchronization, as well as event-related power changes in the gamma range persisted ipsilaterally, although there were differences in temporal and spectral characteristics. These findings support the hypothesis that functional connectivity between hippocampus and rhinal cortex, as well as intrahippocampal connectivity, are partially preserved in hippocampal sclerosis.

Adult↗
PubMed2004

Reduction of human neocortical and guinea pig CA1-neuron A-type currents by organic calcium channel blockers.

In epilepsy models, organic calcium antagonists regularly induce a transient activity increase before suppression of epileptiform discharges. This action was speculated to be mediated by a modulation of potassium currents. Since A-type currents potently regulate neuronal excitability, their modulation by calcium channel blockers was investigated in acutely isolated human neocortical temporal lobe neurons and CA1 neurons of guinea pigs using the whole-cell voltage-clamp technique. In human neurons, 40 microM nifedipine caused an amplitude reduction by 28% at a command potential of -6 mV and produced a biexponential, markedly accelerated current inactivation with time constants of 8.4 +/- 1.1 ms (n = 6) and 62.9 +/- 6.4 ms (n = 5). The time constant under control conditions was 50.1 +/- 8.5 ms (n = 6). Verapamil (40 microM) did not affect the current amplitude, but accelerated the monoexponential current inactivation from 40.2 +/- 7.1 ms to 13.3 +/- 0.8 ms (n = 9). Accordingly, verapamil accelerated the inactivation from 42.3 +/- 5.9 ms to 15.0 +/- 1.3 ms (n = 11) in guinea pig CA1 neurons, without affecting the current amplitude. In this preparation, it was shown that the two enantiomers of verapamil do not differ in their actions. The results show that the A-type current in human neocortical and in guinea pig hippocampal neurons is reduced by organic calcium channel blockers.

Adolescent↗
PubMed2004

Muscarinic acetylcholine receptor stimulation induces expression of the activity-regulated cytoskeleton-associated gene (ARC).

Muscarinic acetylcholine receptors (mAChR) are involved in learning and memory but their molecular function in these processes is not fully understood. In this study, the signal transduction pathway coupling mAChR activation to induction of the activity-regulated cytoskeleton-associated gene (ARC) was examined. ARC was first identified as an effector immediate early gene induced by neuronal activity and ARC protein is thought to play a role in synaptic plasticity. In rats, intraperitoneal injection of pilocarpine, a potent agonist of mAChR, led to increased ARC expression in the brain. In human SH-SY5Y neuroblastoma cells mAChR stimulation with carbachol caused a rapid and robust induction of ARC expression. This effect was inhibited by atropine, a nonselective muscarinic receptor antagonist as well as by M1/M3 subtype-specific antagonists. Analysis of mAChR downstream effectors revealed that protein kinase C (PKC) and tyrosine kinases of the src family are key molecules in the signal cascade leading to ARC expression. Our data suggest, for the first time, that a correlation exists among mAChR-controlled signal cascades, the induction of the effector immediate early gene ARC and synaptic plasticity.

Animals↗
PubMed2004

Increased excitability in cortico-striatal synaptic pathway in a model of paroxysmal dystonia.

Dystonias are movement disorders whose pathomechanism is largely unknown. Dystonic dt(sz) hamsters represent a model of primary dystonias, where alterations of striatal interneuron density and sodium channel function in projection neurones were described. Here, using cortico-striatal slices, we explore whether also the communication between neocortex and striatum is altered in dt(sz) hamsters. Field and intracellular recordings were done in dorsomedial striatum. Electrical stimulation was used to mimic neocortical afferents. Neuronal characteristics, synaptic connections, input-output relations and short- and long-term plasticity were analysed. Regarding cellular properties, striatal neurons of affected animals showed no alterations. Concerning network properties, evoked responses at threshold stimulation were mediated by (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptors. In dt(sz) slices, field responses, paired-pulse accentuation and LTP were larger than in control, possibly by an increase in presynaptic release probability at glutamatergic synapses. In summary, the study indicates that a change of cortico-striatal communication is involved in the manifestation of paroxysmal dystonia in the dt(sz) mutant.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2003

A new neurophysiological/neuropathological ex vivo model localizes the origin of glioma-associated epileptogenesis in the invasion area.

Seizures commonly occur in glioma patients, but their pathogenesis is poorly understood, in part due to a lack of valid and versatile experimental models. We have established a new model that enables comprehensive neuropathological and neurophysiological analysis on identical tissue preparations. Rat C6 glioma cells stably transfected with a green fluorescence protein (GFP) gene are transplanted into rat neocortex, giving rise to diffusely invading gliomas histologically resembling human glioblastomas. After 2 weeks, 500- micro m-thick cerebral slices are prepared, stained with the voltage-sensitive dye RH795, and fluorescence changes associated with origin and spread of abnormal bioelectric activity upon washout of Mg(2+) are detected by a 464-element photodiode array at a rate of 785 frames/s. GFP fluorescence promotes identification of tumor cells during electrophysiological experiments and in neuropathological analyses using frozen and paraffin-embedded tissue sections. By performing subsequent histological analysis of the slices examined neurophysiologically, origin and spread of abnormal activity can be correlated with structural and molecular (immunohistochemical) features. Specifically, we found that ictaform activity was initiated in cortical areas diffusely invaded by single tumor cells. This model is useful for further elucidating the electrophysiological, molecular and structural basis of glioma-associated epileptogenesis.

Animals↗
PubMed2003

Bioelectrical behaviour of hypoxic human neocortical tissue under the influence of nimodipine and dimethyl sulfoxide.

Nimodipine and dimethyl sulfoxide (DMSO) have been shown to affect electrophysiological responses in rodent brain tissue in an vitro model of hypoxia. In the present study, the same agents were now examined for their effects on human neocortical brain slices under repeated hypoxic conditions. DMSO (0.4%), with and without addition of nimodipine (40 micromol/l), did not increase the latency of anoxic depolarization (AD). This finding is not in line with our previous observations of DMSO effects, with and without nimodipine, on brain slices of guinea pigs. AD latency was significantly longer in human neocortical brain slices compared with hippocampal slices of rodents even without any pharmacological influence. A possible acute effect of DMSO-nimodipine may therefore be masked by an interspecies difference of hypoxia resistance.

Action Potentials↗
PubMed2002

Effects of methohexital on bioelectrical reactions in guinea pig hippocampal slices during hypoxia.

The barbiturate methohexital (42 and 140 micromol/l) was tested for an acute effect on anoxic depolarization (AD) and evoked potentials (EP) in hippocampal slices of guinea pigs exposed to repeated hypoxic conditions (n = 78). The dosages of methohexital resemble the range of plasma levels measured in patients with an intraoperative burst suppression electroencephalogram. Direct current potential and EP were recorded in the CA1 region. Hypoxia was terminated either when AD had reached its peak, or 2 min after maximum AD. Excluding the actions of methohexital on cerebral blood flow, reperfusion phase and the delayed mechanisms of cellular protection, a dose-dependent direct effect on EP even after repeated hypoxic conditions could be observed.

Animals↗
PubMed2002

Network and pharmacological mechanisms leading to epileptiform synchronization in the limbic system in vitro.

Seizures in patients presenting with mesial temporal lobe epilepsy result from the interaction among neuronal networks in limbic structures such as the hippocampus, amygdala and entorhinal cortex. Mesial temporal lobe epilepsy, one of the most common forms of partial epilepsy in adulthood, is generally accompanied by a pattern of brain damage known as mesial temporal sclerosis. Limbic seizures can be mimicked in vitro using preparations of combined hippocampus-entorhinal cortex slices perfused with artificial cerebrospinal fluid containing convulsants or nominally zero Mg(2+), in order to produce epileptiform synchronization. Here, we summarize experimental evidence obtained in such slices from rodents. These data indicate that in control animals: (i) prolonged, NMDA receptor-dependent epileptiform discharges, resembling electrographic limbic seizures, originate in the entorhinal cortex from where they propagate to the hippocampus via the perforant path-dentate gyrus route; (ii) the initiation and maintenance of these ictal discharges is paradoxically contributed by GABA (mainly type A) receptor-mediated mechanisms; and (iii) CA3 outputs, which relay a continuous pattern of interictal discharge at approximately 1Hz, control rather than sustain ictal discharge generation in entorhinal cortex. Recent work indicates that such a control is weakened in the pilocarpine model of epilepsy (presumably as a result of CA3 cell damage). In addition, in these experiments electrographic seizure activity spreads directly to the CA1-subiculum regions through the temporoammonic pathway. Studies reviewed here indicate that these changes in network interactions, along with other mechanisms of synaptic plasticity (e.g. axonal sprouting, decreased activation of interneurons, upregulation of bursting neurons) can confer to the epileptic, damaged limbic system, the ability to produce recurrent limbic seizures as seen in patients with mesial temporal lobe epilepsy.

4-Aminopyridine↗