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Cortical spreading depression (CSD) plays a role in migraine with aura. However, studies of the neuronal effects of CSD in human cortex are scarce. Therefore, in the present study, the effects of CSD on the field excitatory postsynaptic potentials (fEPSP) and the induction of long-term potentiation (LTP) were investigated in human neocortical slices obtained during epilepsy surgery. CSD significantly enhanced the amplitude of fEPSP following a transient suppressive period and increased the induction of LTP in the third layer of neocortical tissues. These results indicate that CSD facilitates synaptic excitability and efficacy in human neocortical tissues, which can be assumed to contribute to hyperexcitability of neocortical tissues in patients suffering from migraine.

Eugenol, an aromatic molecule derived from several plants, has been receiving examination for clinical relevance in epilepsy and headache. To investigate the neurophysiologic properties of the action of eugenol, its effects on epileptiform field potentials elicited by omission of extracellular Mg2+, spreading depression induced by KCl microinjection, electrically evoked field potentials, and long-term potentiation were tested in rat neocortical and hippocampal tissues. Eugenol (10-100 micromol/l) dose-dependently and reversibly suppressed both epileptiform field potentials and spreading depression Eugenol also reversibly decreased the amplitude of the field postsynaptic potentials evoked in CA1 area of hippocampus and the third layer of neocortex. Eugenol significantly reduced the long-term potentiation by approximately 30% compared with controls. Thus, eugenol can suppress epileptiform field potentials and spreading depression, likely via inhibition of synaptic plasticity. The results indicate the potential for eugenol to use in the treatment of epilepsy and cephalic pain.

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 microM 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 microM 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., (Copyright 2004 Wiley-Liss, Inc.)

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.

Spreading depression (SD) is characterized by a transient breakdown of neuronal function concomitant with a massive failure of ion homeostasis. It is a phenomenon that can be induced in neocortical tissue by raising excitability, e.g. injection of K(+), application of glutamatergic agonists, or blocking Na(+)/K(+) ATPase. Here we report a novel method of SD induction using minimal disinhibition with application of low concentrations (5 microM) of the GABA(A) receptor blocker bicuculline. This procedure-while subthreshold for epileptiform activity-readily induced spontaneous SDs in native rat neocortical slices, accompanied by typical depolarizations of neurons and glial cells. In contrast, in human neocortical preparations obtained from epilepsy surgery, in approximately 20% of the slices spontaneous epileptiform activity appeared with this bicuculline dosage without SDs. Raising the concentration of bicuculline to an epileptogenic dose (10 microM) in human tissue also resulted in the generation of epileptiform activity only. Likewise, in slices from pilocarpine-treated, chronically epileptic rats, bicuculline also only induced epileptiform activity without eliciting SDs. The experiments indicate that chronic epilepsy causes a differential sensitivity to partial GABA(A) receptor blockade with regard to induction of SD.

Stimulus-induced pattern of bioelectric activity in human neocortical tissue was investigated by use of the voltage sensitive dye RH795 and a fast optical recording system. During control conditions stimulation of layer I evoked activity predominantly in supragranular layers showing a spatial extent of up to 3000 microm along layer III. Stimulation in white matter evoked distinct activity in infragranular layers with a spatial extent of up to 3000 microm measured along layer V. The mean amplitude of optical signals close to the stimulated sites in layer I and white matter determined 25 ms following the stimulus, decreased by 50% at a lateral distance of approximately 900 microm and 1200 microm, respectively. Velocity of spread along the vertical stimulation axis reached 0.24 m/s in the supragranular layers (layers I to III) and then decreased to 0.09 m/s following layer I activation; stimulation of white matter induced a velocity of spread in layer V of 0.38 m/s, which slowed down to 0.12 m/s when passing the lower border of lamina IV. The horizontal velocities of spread determined from the stimulation site to a lateral distance of 500 microm reached 0.26-0.28 m/s and 0.28-0.35 m/s for layer I and white matter stimulation, respectively. At larger distances velocity of spread decreased. Increased excitability (Mg(2+)-free solution) had no significant effect on the spatio-temporal distribution of evoked activity as compared with control conditions. There were also no obvious differences between the results obtained in slices, which generated spontaneously sharp waves and those which were not spontaneously active. About 30% of the slices (n=7) displayed a greatly different response pattern, which seemed not to be related in a simple way to the stimulation as was the case in the majority of the investigated slices. The activity pattern of those slices appeared atypical in regard to their deviations of the vertical and horizontal extent of activity, to their reduced spatial extent of activity during increased excitability, to their layer-related distribution of activity, and to the appearance of afterdischarges.Concluding, in 30% of the human temporal lobe slices atypical activity pattern occurred which obviously reflect intrinsic epileptiform properties of the resected tissue. The majority of slices showed stereotyped activity pattern without evidence for increased excitability.

Optical imaging of neuronal network activity yields information of spatial dynamics which generally is analyzed visually. The transient appearance of spatial activity patterns is difficult to gauge in a quantifiable manner, or may even altogether escape detection. Here, we employ geometric shape matching using Fréchet distances or straight skeletons to search for pre-selected patterns in optical imaging data with adjustable degrees of tolerance. Data were sampled from fluorescence changes of a voltage-sensitive dye recorded with a 464-photodiode array. Fluorescence was monitored in a neuronal network in vitro. Neuronal activity prompting fluorescence fluctuations consisted of spontaneous epileptiform discharges in neocortical slices from patients undergoing epilepsy surgery. The experiments show that: (a) spatial activity patterns can be detected in optical imaging data; (b) shapes such as "mini-foci" appear in close correlation to bioelectric discharges monitored with field potential electrodes in a reproducible manner; (c) Fréchet distances yield more conservative matches regarding rectangular, and less conservative hits with respect to radially symmetric shapes than the straight skeleton approach; and (d) tolerances of 0.03-0.1 are suited to detect faithful images of pre-selected shapes, whereas values >0.8 will report matches with any polygonal pattern. In conclusion, the methods reported here are suited to detect and analyze spatial, geometric dynamics in optical imaging data.

Cortical spreading depression (CSD) occurrence has been suggested to be associated with seizures, migraine aura, head injury and brain ischemia-infarction. Only few studies identified CSD in human neocortical slices and no comprehensive study so far evaluated this phenomenon in human. Using the neocortical tissue excised for treatment of intractable epilepsy, we aimed to investigate CSD in human. CSD was induced by KCl injection and by modulating T-type Ca(2+) currents in incubated human neocortical tissues in an interphase mode. The DC-fluctuations were recorded by inserting microelectrodes into different cortical layers. Local injection of KCl triggered single CSD that propagated at 3.1+/-0.1 mm/min. Repetitive CSD also occurred spontaneously during long lasting application (5 h) of the T-type Ca(2+) channel blockers amiloride (50 microM) or NiCl(2) (10 microM) which was concomitant with a reversible extracellular potassium increase up to 50 mM. CSD could be blocked by the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonovaleric acid in all cases. The results demonstrate that modulation of the Ca(2+) dynamics conditioned human neocortical slices and increased their susceptibility to generate CSD. Furthermore, these data indicate that glutamatergic pathway plays a role in CSD phenomenon in human.

Functional changes in neuronal circuitry reflected in spontaneously occurring synchronous sharp field potentials (SSFP) have been reported to occur in human brain suffering from chronic epileptogenicity but not in primary nonepileptic tissue from peritumoral resectates. Voltage sensitive dyes and fast imaging were used to visualize spontaneously occurring rhythmic depolarizations correlated to SSFP in chronically epileptic human neocortical slices obtained during epilepsy surgery. Localized and spatially inhomogeneous neuronal depolarizations were found to underlie spontaneous SSFP, which remained unchanged and spatially restricted to foci <750 micrometer diam even under epileptogenic (low-Mg(2+)) conditions. In cases where ictaform paroxysmal activity occurred in low-Mg(2+) medium, neuronal depolarizations were wide-spread but still spatially inhomogeneous, and the events were preferentially initiated at distinct foci. The findings suggest that small neuronal networks are able to establish and maintain synchronous rhythmic and epileptiform activity.

It has been demonstrated in animal models that chronic epilepsy is associated with increased excitability which may result from abnormal glutamatergic transmission involving altered properties of N-methyl-D-aspartate (NMDA) receptors. We have investigated whether human temporal lobe epilepsy is associated with changes in the NMDA receptor at the molecular level by assessing the relative expression of mRNAs of the different splice variants at the N-terminal (exon 5) and C-terminal (exon 21) position for the NMDAR1 subunit. Specimens of hippocampus and temporal lobe cortex from patients with refractory epilepsy were obtained during neurosurgical operations and analyzed by means of the reverse transcription reaction followed by polymerase chain reaction. Non-epileptic control specimens obtained at autopsy exhibited a relatively high level in expression of exon 5-lacking (hippocampus: 0.87; cortex: 0.81) and exon 21-containing (hippocampus: 0.95; cortex: 0.93) transcripts. The ratio for these alternatively spliced transcripts was not significantly changed in epileptic hippocampal and cortical tissues relative to the corresponding non-epileptic samples. These results did not support a potential role for NMDAR1 splice variants in the pathophysiology of epilepsy.

The antiepileptic effect of the dihydropyridine calcium channel blocker nifedipine was tested in neocortical slice preparations (n=27) from patients ranging in age from four to 46 years (mean=25) who underwent surgery for the treatment of intractable epilepsy. Epileptiform events consisted of spontaneously occurring rhythmic sharp waves as well as of untriggered epileptiform field potentials induced by omission of Mg(2+) from the superfusate, or epileptiform field potentials elicited by application of bicuculline and triggered by single electrical stimuli. (1) Spontaneous rhythmic sharp waves (n=6): with nifedipine (40micromol/l), the repetition rate was decreased down to 30% of initial value, whereas the area under the field potential remained nearly unchanged. (2) Untriggered low Mg(2+) epileptiform field potentials (n=6): with nifedipine (40micromol/l) the area under the field potentials was reduced while the action on the repetition rate was ambiguous. (3) Triggered bicuculline epileptiform field potentials (n=15): with nifedipine (40micromol/l; n=4), no antiepileptic effect was found. There was, however, a marked increase in the area under the epileptiform field potentials. The area under the field potentials was reduced only at a dosage of 60micromol/l (n=11). This effect was stronger when nifedipine was applied with a K(+) concentration raised from 4 to 8mmol/l. The results show that the calcium channel blocker nifedipine is able to reduce differential epileptiform discharges in human neocortical tissue. These observations are in line with previous findings, suggesting that calcium flux into neurons is involved in epileptogenesis. The present results therefore support the idea that some organic calcium antagonists may be useful in human epilepsy therapy, although the etiology of epileptic seizures seems to be a critical factor for the efficacy of the drug.

The aim of the present investigation was to compare the antiepileptic efficacy of the specific L-type calcium channel blocker nifedipine in hippocampal and neocortical slice preparations in the Mg2+-free model of epilepsy. The main findings were as follows. (1) In hippocampal slices, in general, nifedipine (20-80 micromol/l) exerted a suppressive effect both on repetition rate and on area under epileptiform field potentials. This effect was clearly dose dependent. In the majority of cases, this suppression was preceded by an increase, which was transient in nature. Only in the lowest concentration (20 micromol/l) used, in normal K+, instead of a depression, a persistent increase occurred. (2) In neocortical slices, in the majority of experiments, nifedipine (20-80 micromol/l) showed a depressive action only on the area under the epileptiform field potentials. The depressive effect of nifedipine on the area was dose dependent, although to a lesser extent than in the hippocampus. In nearly half of the slices this suppression was preceded by a transient increase. By contrast, the repetition rate of epileptiform field potentials increased transiently in about 20% of the slices followed by a decrease. In the remaining 80% of the slices the repetition rate increased persistently. (3) An elevation of the K+ concentration accentuated the depressive actions of nifedipine only in the hippocampus. In contrast to elevated K+, in both the hippocampus and the neocortex, epileptiform field potentials were not suppressed in all experiments in normal K+. (4) The reversibility of the depressive effects of nifedipine was differential in the two tissue types. In the hippocampus, after suppression of epileptiform field potentials they reappeared in the overwhelming majority of slices. In the neocortex, this was the case in only one experiment. These findings may indicate the existence of L-type calcium channels with a differential functional significance for epileptogenesis and/or the existence of different forms of L-type channels in hippocampal and neocortical tissue. As a whole, the differential effects of L-type calcium channel blockade in the hippocampus and neocortex point to differences in the network properties of the two tissue types.

In human neocortical slices obtained during epilepsy surgery, sharp waves have been described to appear spontaneously, the shape of which met all criteria of epileptiform field potentials. In the present investigation, the current sinks and sources underlying these potentials were analysed. The cortical tissue used in the present study was a small portion of the tissue blocks excised for treatment of pharmacoresistant focal epilepsy. The tissue came from the temporal (n = 26), frontal (n = 1) and parietal (n = 1) lobes. Slices of 500 microm thickness were cut in the frontal plane perpendicular to the pial surface. Field potentials were recorded using a linear array of eight wire electrodes (diameter: 33 microm) with interelectrode distances of 300 microm. To scan a slice for sharp field potentials, this array was placed perpendicular to the pial surface at the midsection of each preparation, and consecutively at the respective midsections of the resulting halves of the slice. Each of these locations was termed a recording line. Depending on the appearance of spontaneous potentials, recording lines and slices were classified as either spontaneous or non-spontaneous. With both spontaneous and zero Mg(2+)-induced interictal discharges, in spontaneous slices, current sinks were preferentially located in layers II and III. In non-spontaneous slices, current sinks associated with interictal potentials could be found throughout all cortical laminae. With zero Mg(2+)-induced ictal activity, in spontaneous slices, the initial sinks were preferentially located in cortical laminae II and IIIa, and were shifted to lower ones after additional application of bicuculline. In non-spontaneous slices, no ictal-type discharges could be induced with omission of Mg2+ from the superfusate. Only addition of bicuculline elicited ictal-type activity, and sinks associated with this were preferentially located in layers II and IIIa. The results suggest that the supragranular layers, especially layer II, change qualitatively in functional organization in slices showing spontaneous discharges. We think that this special feature represents the function of the upper layers and can be blocked by bicuculline. This interpretation is supported by the observation that ictal discharges normally started in the upper layers in spontaneous and non-spontaneous slices, except for spontaneous slices with bicuculline, where the zone initiating discharges was translocated to deeper layers.

Since a disturbed balance between excitatory and inhibitory amino acid receptors is suggested to be an important condition for epileptogenic cortical activity, the present study has focused on the analysis of the densities of (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), N-methyl-D-aspartate, kainate and GABA subtype A receptors in neocortical tissue surgically removed from patients with focal epilepsy. The mean densities (collapsed over cortical layers I-VI) and the laminar distribution patterns of [3H]AMPA, [3H]MK-801, [3H]kainate and [3H]muscimol binding to AMPA, N-methyl-D-aspartate, kainate and GABAA receptors were determined with quantitative receptor autoradiography in the neocortex of patients with focal epilepsy and controls. The tissue probes used in the present study were functionally characterized by parallel electrophysiological investigations. From that, the different probes could be subdivided into a spontaneously spiking and a non-spontaneously spiking group. The mean density of [3H]AMPA binding sites was significantly increased (+37%) in the group of epileptic brains (n = 10) compared with controls (n = 10), but the mean densities of [3H]MK-801, [3H]kainate and [3H]muscimol binding sites were not significantly altered (-8%, +/-0% and -7%, respectively). The relation between the densities of all four binding sites were simultaneously displayed as polar plots in each single brain ("receptor fingerprints"). The consistent up-regulation of [3H]AMPA binding sites in all epileptic brains was found to be associated with a down-regulation of the N-methyl-D-aspartate receptor in four of the five non-spontaneously spiking cases, and an associated up-regulation of the N-methyl-D-aspartate receptor was seen in all spontaneously spiking cases. Finally, the laminar distribution of binding site densities was analysed, since the mean densities collapsed over all neocortical layers may obscure layer-specific alterations. Layer- and receptor- specific up- or down-regulations were found in epileptic tissue compared with controls. Moreover, the laminar distribution pattern of current sinks associated with epileptiform potentials in a spontaneously spiking cortical slice was found to be co-localized with local maxima of AMPA receptor densities. The present analysis of four ionotropic glutamate and GABA receptor subtypes demonstrates a consistent and significant up-regulation of [3H]AMPA binding sites in all cases of human focal epilepsy, which co-localizes with the occurrence of sinks in current-source-density analysis. The receptor fingerprint analysis suggests a subdivision of focal epilepsy into two subtypes on the basis of neurochemical/functional correlations: (i) a spontaneously spiking subtype with increased N-methyl-D-aspartate receptor density, and (ii) a non-spontaneously spiking subtype with decreased N-methyl-D-aspartate receptor density.

The spatio-temporal distribution of epileptiform activity was investigated in slices from human temporal neocortex resected during epilepsy surgery. Activity was recorded by use of a voltage-sensitive dye and an optical recording system. Epileptiform activity was induced with 10 microM bicuculline and electrical stimulation of layer I. In 10 slices from six patients investigated, epileptiform activity spread across most of the slice. Largest amplitudes were located in layer II/III. Epileptiform activity was characterized by long-lasting potentials with slow rising phases and a low velocity of spread in the horizontal direction (0.044 m/s). This spatio-temporal pattern of epileptiform activity in human slices was similar to that found previously in neocortical slices from guinea pigs with bicuculline. In four of nine human slices investigated under control bath conditions (in non-epileptogenic medium), the spatio-temporal activity patterns were similar to those of guinea pigs in non-epileptogenic medium. In the remaining five human slices, however, the spread in the horizontal direction was significantly larger (4188 microm) in non-epileptogenic medium than that found in slices from guinea pigs (2171 microm). Activity in human slices showing such 'wide spread' in control bath conditions occasionally had characteristic features of epileptiform activity. Further work will have to clarify whether these epileptiform features reflect intrinsic epileptiform properties in human tissue slices.

Alongside GABA, glycine is the major inhibitory transmitter in the central nervous system. Application of the glycine receptor blocker strychnine is known to evoke epileptiform phenomena. The present paper addresses the question whether postsynaptic calcium currents through L-type channels contribute to strychnine-induced epileptiform field potentials (EFP). To test for this, the antiepileptic effect of the specific L-type calcium channel blocker, verapamil, in hippocampal and neocortical slices was investigated. In parallel with this, the antiepileptic efficacy of the unspecific calcium channel modulator, flunarizine, was tested with respect to pharmacotherapy of epilepsies. In both preparations, the L-type calcium channel blocker, verapamil, was able to suppress EFP. In neocortical slices, EFP were blocked in all experiments, whereas in hippocampal slices, in 3 out of 11 experiments, no complete suppression occurred. Flunarizine acted in a similar way. It is concluded that L-type calcium channels are involved in strychnine-induced epilepsy, but to a greater extent in the neocortex than in the hippocampus.

Growing numbers of evidence indicate that cognitive impairments are part of clinical profile of childhood absence epilepsy. Little is known on neuropathological changes accompanied by cognitive deficits in absence epilepsy. The aim of the present study was to investigate age-dependent neuropathological changes accompanied by learning and memory impairments in Wistar Albino Glaxo from Rijswijk (WAG/Rij) rat model of absence epilepsy. Experimental groups were divided into four groups of six rats of both WAG/Rij and Wistar strains with 2 and 6 months of age. The learning and memory performances were assessed using passive avoidance paradigm and neuropathological alterations were investigated by the evaluation of the number of dark neurons and apoptotic cells as well as the expression of caspase-3 in the neocortex, the hippocampus, and different regions of the thalamus. Results revealed a decline in learning and spatial memory of 6-month-old WAG/Rij rats compared to age-matched Wistar rats as well as 2-month-old WAG/Rij and Wistar rats. The mean number of dark neurons was significantly higher in the hippocampal CA1 and CA3 areas as well as in the laterodorsal, centromedial, and reticular thalamic nuclei and the somatosensory cortex of 6-month-old WAG/Rij rats. In addition, a higher number of apoptotic cells as well as a higher expression of caspase-3 was observed in the hippocampal CA1 and CA3 regions, the laterodorsal thalamic nucleus, and the somatosensory cortex of 6-month-old WAG/Rij rats compared to other animal groups. These results indicate significant enhancement of neuronal damage and cell death accompanied by memory deficits after seizure attacks in a rat model of absence epilepsy. Seizure-induced neuronal injury and death may underlie cognitive impairments in absence epilepsy. [ABSTRACT FROM AUTHOR]

Optical imaging of neuronal network activity yields information of spatial dynamics which generally is analyzed visually. The transient appearance of spatial activity patterns is difficult to gauge in a quantifiable manner, or may even altogether escape detection. Here, we employ geometric shape matching using Fre´chet distances or straight skeletons to search for pre-selected patterns in optical imaging data with adjustable degrees of tolerance. Data were sampled from fluorescence changes of a voltage-sensitive dye recorded with a 464-photodiode array. Fluorescence was monitored in a neuronal network in vitro. Neuronal activity prompting fluorescence fluctuations consisted of spontaneous epileptiform discharges in neocortical slices from patients undergoing epilepsy surgery. The experiments show that: (a) spatial activity patterns can be detected in optical imaging data; (b) shapes such as ‘mini-foci’ appear in close correlation to bioelectric discharges monitored with field potential electrodes in a reproducible manner; (c) Fre´chet distances yield more conservative matches regarding rectangular, and less conservative hits with respect to radially symmetric shapes than the straight skeleton approach; and (d) tolerances of 0.03–0.1 are suited to detect faithful images of pre-selected shapes, whereas values >0.8 will report matches with any polygonal pattern. In conclusion, the methods reported here are suited to detect and analyze spatial, geometric dynamics in optical imaging data. [Copyright &y& Elsevier]