Rüdiger Köhling

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.

RNA editing (R/G site) and flip-flop splicing of the AMPA receptor subunit GluR2 in nervous tissue of epilepsy patients

Editing and alternative splicing of mRNA are posttranscriptional steps probably involved in pathophysiological aspects of epilepsy. The present study analyses the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit GluR2 with respect to the expression of (i) editing at the R/G site and (ii) flip-flop cassettes. Nervous tissue from patients with temporal lobe epilepsy was analysed by RT-PCR followed by restriction enzyme assays. Human autoptic tissue served as control. R/G editing status: the relative amount of edited RNA was significantly increased in the hippocampal tissue, whereas no changes were found in neocortical tissues. Flip-flop expression: no significant alterations were found in relative abundance of spliced variants containing the flip exon. The increased editing at the R/G site in the hippocampal tissue of epilepsy patients may enhance responses to glutamate, resulting in a synapse operating at an increased gain.

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.

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.

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 2xa0weeks, 500-µm-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 Mg2+ 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.

Differential involvement of L-type calcium channels in epileptogenesis of rat hippocampal slices during ontogenesis.

Organic calcium channel antagonists block epileptiform activity in adult tissue, suggesting an essential role of L-type channels in epileptogenesis in the mature CNS. By contrast, this remains doubtful for neonatal tissue, as the density of calcium channels changes markedly with ontogenesis. The paper addresses this question by exploring the antiepileptic efficacy of the L-type calcium channel blockers verapamil and nifedipine in low-Mg(2+)-epilepsy in rat hippocampal slices of different postnatal (PN) ages. Field (CA3, CA1) and membrane potentials (CA3) were recorded. Washout of Mg(2+) induced epileptiform potentials, which were blocked age-dependently: Verapamil suppressed activity in all preparations of PN1-5 and PN13-30+, but only in 70% of PN6-12. Nifedipine depressed activity in >75% of slices of PN13-30+, but only in 33% of PN1-12. The findings indicate a role of L-type calcium channels in epileptogenesis from PN13 onwards, with phenylalkylamine-sensitive calcium channels also being involved during PN1-5.

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.

Characterization of a fast transient outward current in neocortical neurons from epilepsy patients.

A‐type currents powerfully modulate discharge behavior and have been described in a large number of different species and cell types. However, data on A‐type currents in human brain tissue are scarce. Here we have examined the properties of a fast transient outward current in acutely dissociated human neocortical neurons from the temporal lobe of epilepsy patients by using the whole‐cell voltage‐clamp technique. The A‐type current was isolated with a subtraction protocol. In addition, delayed potassium currents were reduced pharmacologically with 10 mM tetraethylammonium chloride. The current displayed an activation threshold of about −70 mV. The voltage‐dependent activation was fitted with a Boltzmann function, with a half‐maximal conductance at −14.8 ± 1.8 mV (n = 5) and a slope factor of 17.0 ± 0.5 mV (n = 5). The voltage of half‐maximal steady‐state inactivation was −98.9 ± 8.3 mV (n = 5), with a slope factor of −6.6 ± 1.9 mV (n = 5). Recovery from inactivation could be fitted monoexponentially with a time constant of 18.2 ± 7.5 msec (n = 5). At a command potential of +30 mV, application of 5 mM 4‐aminopyridine or 100 μM flecainide resulted in a reduction of A‐type current amplitude by 35% or 22%, respectively. In addition, flecainide markedly accelerated inactivation. Current amplitude was reduced by 31% with application of 500 μM cadmium. All drug effects were reversible. In conclusion, neocortical neurons from epilepsy patients express an A‐type current with properties similar to those described for animal tissues.

Sodium Currents in Striatal Neurons from Dystonic dtsz Hamsters: Altered Response to Lamotrigine

Dystonic mutant dt(sz) hamsters are a model for paroxysmal dystonia. Handling/stress provoke the dystonic attacks. This phenomenon subsedes with maturation, but can be reinvoked when these animals receive sodium channel blockers such as lamotrigine, suggesting a dysfunction of striatal sodium channels. Voltage-gated fast sodium currents (I(Na(+))) were studied in acutely isolated striatal neurons from healthy and dt(sz) hamsters in whole-cell voltage clamp recordings. The action of lamotrigine was tested on (a) current/voltage relationship, (b) kinetics, and (c) steady-state inactivation and activation. Under control conditions, properties of I(Na(+)) were not different between healthy and dt(sz) neurons. With lamotrigine, however, (a) peak currents were significantly less depressed by the drug in neurons from dt(sz) hamsters as compared to healthy cells, and (b) the steady-state inactivation curve shift of I(Na(+)) was less pronounced in dt(sz) neurons. The results suggest that in dt(sz) hamsters, fast sodium currents in striatal neurons are more resistant to blockade. This sodium channel alteration might be causal for a functional imbalance between input and output structures of the basal ganglia under conditions of compromised I(+)(Na).

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.