Comparison of spontaneous and evoked epileptiform activity in three in vitro epilepsy models.

Rat neocortical slices express spontaneous epileptiform activity after incubation with GABA(A) receptor blocker bicuculline (BIC, 20 microM), with potassium channel blocker 4-aminopyridine (4-AP, 50 microM) or in Mg(2+)-free medium (LMG). Various parameters of spontaneous and evoked epileptiform discharges and their pharmacological sensitivity were analysed using extracellular field potential recordings in this comparative in vitro study. All types of convulsant solution induced spontaneous epileptiform activity, however, the analysed parameters showed that characteristics of epileptiform discharges are rather different in the three models. The longest duration of discharges was recorded in LMG, while the highest frequency of spontaneous events was detected in 4-AP. The epileptiform field responses elicited by electrical stimulation appeared in an all-or-none manner in BIC. On the contrary, in 4-AP and in LMG the amplitude of the responses increased gradually with increasing stimulation intensities. The NMDA receptor antagonist D,L-2-amino-5-phosphonovaleric acid (APV, 25 microM) abolished the LMG induced spontaneous epileptiform activity and significantly reduced the frequency of the epileptiform discharges in BIC and 4-AP. Blocking the AMPA type of glutamate transmission with 1-(aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466, 40 microM) rapidly abolished BIC-induced spontaneous epileptiform activity and caused a significant decrease in the frequency of 4-AP induced spontaneous epileptiform discharges. However, it had only a weak effect on the LMG-induced epileptiform activity. We conclude that the contribution of NMDA and AMPA types of glutamate receptors to the development and maintenance of epileptiform activity in cortical cell assemblies is different in the three models. There are significant alterations in contribution of NMDA and AMPA types of glutamate receptors to the above-mentioned processes in the different convulsants. In BIC the synchronisation is mainly due to the altered network properties, namely inhibition is reduced in the local circuits. Although inhibition is reduced in the local circuits, the AMPA receptor antagonist relatively easily blocked the synchronised excitation. In 4-AP, and especially in LMG, changes in the membrane characteristics of neurones play a crucial role in the increased excitability. In this case the AMPA antagonist was less effective.

Altered electrophysiological characteristics of developing rat cortical neurones after chronic methylmercury chloride treatment.

The effect of methylmercury chloride on the excitability of developing cortical neurons was tested. Methylmercury was administered in the drinking water to pregnant rats during gestation and suckling period and the offspring were investigated. The electrical characteristics of the neuronal membranes as well as the synaptic responses evoked by electrical stimulation of the corpus callosum were measured in brain slices. Slices prepared from the somatosensory cortex of 4-week-old rats were analyzed using sharp electrode intracellular microelectrophysiological recording technique. Long-lasting treatment with low doses of methylmercury chloride caused a slight decrease in the membrane potential and in the amplitude of spikes together with an enhanced excitability. Some of the treated animals were grown up without any further methylmercury application, and their offspring (second generation) were also studied electrophysiologically. These untreated offspring had normal neuronal characteristics. The altered membrane characteristics detected in the 4-week-old MeHg treated animals might be the consequence of the abnormal developmental processes taking place in the presence of MeHg which may alter the normal neuronal excitability. Besides this, the acute toxic effect of Hg(2+), which was present in the brain at the time of investigations, has to be taken into consideration.

An in vitro electrophysiological and Co2+-uptake study on the effect of infraorbital nerve transection on the cortical and thalamic neuronal activity.

Changes of neuronal membrane characteristics in somatosensory barrel cortex and barreloid thalamus were investigated in rats following unilateral transection of the infraorbital nerve. Kainate induced Co2+-uptake method and image analysis were used to assess the Ca2+ permeability of non-NMDA (N-methyl-D-aspartate) glutamate receptors. Changes in some biophysical parameters of the affected cortical neurons were also investigated by intracellular recording in slice experiments. The altered neuronal activity was measured on days 1, 5 and 14 after surgery. Kainate induced Co2+ uptake increased markedly reflecting enhanced Ca2+ permeability of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate/kainate (AMPA/KAIN)-type receptors. Changes were more pronounced in the cortex than in the thalamus and peaked on the first day following nerve transection. After that, parameters gradually returned to the normal level. However, a small enhancement was still detectable in the cortex at the end of the 2-week-long observation period. In parallel with the increased Co2+-uptake, moderate membrane potential changes, stronger spiking activity and enhanced excitability were characteristic for cortical neurons. The observed alterations in neuronal characteristics underlie the reorganization and regeneration processes following injuries or surgeries. We can conclude that immediate change of the receptive field in the barrel cortex following unilateral nerve transection is based on changes in biophysical parameters of the neurons. Altered peripheral activation evokes changes in the neuronal activity, thus providing opportunity for a quick synaptic rearrangement. AMPA/KAIN-type glutamate receptors have a decisive role in the regulation of these processes. This kind of synaptic plasticity is more significant in the cortex than in the thalamus.

Effect of a glutamate receptor antagonist (GYKI 52466) on 4-aminopyridine-induced seizure activity developed in rat cortical slices.

In the present experiments we have tested the effect of the noncompetitive AMPA antagonist GYKI 52466 (20-80 microM) on spontaneous epileptic discharges developed as the consequence of 4-aminopyridine application in neocortex slices of adult rats. Parallel to the changes of spontaneous activity, the field potentials, evoked by electrical stimulation of the corpus callosum, were also analyzed. Glass microcapillary extracellular recording electrode was positioned in the third layer of the somatosensory cortex slice, while the stimulating electrode was placed at the border of the white and gray matter. 4-aminopyridine and GYKI 52466 were bath-applied. The application of 40 microM GYKI 52466 caused about 40% decrease in the frequency and the amplitude of spontaneous seizures as well as the duration of each discharges developed in 4-amino-pyridine. Pre-incubation with the AMPA antagonist effectively inhibited both the development of seizure activity and the maintenance of the discharges. GYKI 52466 also decreased the duration and amplitude of field responses evoked by stimulation of the corpus callosum. This inhibitory effect was dose-dependent. Our data in the in vitro cortex slice epilepsy model suggest that the non-competitive AMPA antagonist GYKI 52466 is a potent anticonvulsant and neuroprotective compound because it reduced the fully developed epileptic discharges or prevented their development.

Effect of glutamate receptor antagonists on excitatory postsynaptic potentials in striatum.

Glutamate, as the main transmitter of corticostriatal pathway, has a crucial role in the regulation of the activity of striatal cells as well as in pathogenesis of some diseases characterized by striatal malfunction caused by overexcitation of neurons. In the present study, the role of ionotropic excitatory amino acid receptors was investigated in the striatal synaptic transmission. Using conventional intracellular electrophysiological methods in brain slices, we have investigated the effects of the N-methyl-D-aspartate (NMDA) antagonist (+/-) 2-amino-5-phosphono-valerate (APV) and the alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) antagonist (+/-) 1-(4-aminophenyl)-3-methyl-carbamoyl-7,8-methylenedioxy-5H-2,3-benzodiaz epine (GYKI 53655) on the excitatory postsynaptic potentials (EPSPs) evoked by electrical stimulation of corpus callosalpham. The AMPA antagonist significantly decreased electrically evoked responses and a weak inhibition was also observed after APV application. The results were compared to similar data obtained in a cortical slice study.

An increased intensity of N-methyl-D-aspartate (NMDA) but not non-NMDA receptor activation may be responsible for the enhancement of excitatory processes in the neocortex of two-week-old rats: a brain slices study.

During the brain maturation a critical period is detectable when the sensitivity of the neocortex is high. Enhanced excitatory activity is characteristic at that time while the inhibitory processes are underdeveloped. The goal of this study was to determine the effectiveness of different types of excitatory amino acid antagonists reducing the electrically evoked excitatory synaptic responses of the somatosensory cortex. Effects of the specific competitive N-methyl-D-aspartate (NMDA) antagonist 4-amino-phosphono-valerate (APV), and the specific non-competitive, non-NMDA antagonist 1-(4-aminophenyl)-3-methylcarbamoyl-7,8-methylenedioxy-5H-2,3-benz odiazepine (GYKI 53655) were analysed on neocortex slices prepared from 2-week-old and adult rats. APV caused a partial inhibition of the electrically evoked response more effectively in young animals than in adults, while the effective IC50 values were similar. In contrast, the non-NMDA antagonist had a similar effect on the slices of both age-groups.

Low extracellular magnesium unmasks N-methyl-D-aspartate-mediated graft-host connections in rat neocortex slice preparation.

The main purpose of this study was to investigate the role of N-methyl-D-aspartate receptors in host-graft synaptic transmission in the neocortex. The effects of low extracellular magnesium, the glutamate agonist N-methyl-D-aspartate and N-methyl-D-aspartate antagonists on the synaptic activation of connections between embryonic neocortical graft tissue and the surrounding host tissue were studied in 17 perfused slices of rat neocortex. In standard artificial cerebrospinal fluid, stimulation of the host white matter evoked field potentials in four of 17 grafts. However, in Mg(2+)-free medium, the same stimulation evoked field potentials in an additional six grafts, with significant increases in the mean duration of the evoked responses in the 10 responsive grafts. In five of these slices stimulation of the graft also evoked field potentials in the host tissue, suggesting reciprocal interaction between graft and host. Simultaneous extracellular recordings from graft and host tissues in Mg(2+)-free medium showed that spontaneous epileptiform discharges developed in the graft and host tissue synchronously. In Mg(2+)-free medium, application of N-methyl-D-aspartate induced a shift of the baseline with superimposed epileptiform discharges in both graft and host. Application of the non-competitive N-methyl-D-aspartate antagonist ketamine and the competitive antagonist D,L-2-amino-5-phosphonovaleric acid attenuated or reversibly blocked both the spontaneous epileptiform discharges and the evoked field potentials. Our data provides evidence that N-methyl-D-aspartate receptors are present at synapses created between fetal graft and host neocortex, and that the N-methyl-D-aspartate-activated receptor-channel complex plays an active role in mediating excitatory synaptic transmission in host-graft circuitry.

Development of long-term potentiation in the somatosensory cortex of rats of different ages.

The age dependence of possible long-term potentiation (LTP) induction in rat somatosensory cortex was studied in in vitro slice experiments. Coronal slices were prepared from the somatosensory cortex of rats of different ages, and excitatory postsynaptic potentials evoked by stimulation of the white matter (0.1 Hz, subthreshold for spike) were recorded intracellularly. In 70% of the slices taken from 2-week-old rats, a moderate potentiation (20-30%) could be induced by either 5 or 100 Hz stimulation. No LTP was observed in younger (1 week) or older (3 weeks) cortex. On the basis of our experiments an important ontogenetic role of increased synaptic efficacy is suggested in a critical developmental period of rats after birth.

Changes in seizure activity of the neocortex during the early postnatal development of the rat: an electrophysiological study on slices in Mg(2+)-free medium.

NMDA receptor-mediated process of rat neocortex slices prepared from 2-24-day-old rats were studied in Mg(2+)-free solution. The response to NMDA application as well as the susceptibility to epileptiform discharges showed age-dependent changes during the first 4 weeks. Slices from the youngest age group seemed to be the most sensitive to NMDA, whereas epileptic activity developed most readily at around the third week.