Effect of endothelin-1 on the excitability of rat cortical and hippocampal slices in vitro.

Endothelin-1 (ET-1) is a neuroactive protein produced in most brain cell types and participates in regulation of cerebral blood flow and blood pressure. In addition to its vascular effects, ET-1 affects synaptic and nonsynaptic neuronal and glial functions. Direct application of ET-1 to the hippocampus of immature rats results in cerebral ischemia, acute seizures, and epileptogenesis. Here, we investigated whether ET-1 itself modifies the excitability of hippocampal and cortical circuitry and whether acute seizures observed in vivo are due to nonvascular actions of ET-1. We used acute hippocampal and cortical slices that were preincubated with ET-1 (20 µM) for electrophysiological recordings. None of the slices preincubated with ET-1 exhibited spontaneous epileptic activity. The slope of the stimulus intensity-evoked response (input-output) curve and shape of the evoked response did not differ between ET-1-pretreated and control groups, suggesting no changes in excitability after ET-1 treatment. The threshold for eliciting an evoked response was not significantly increased in either hippocampal or cortical regions when pretreated with ET-1. Our data suggest that acute seizures after intrahippocampal application of ET-1 in rats are likely caused by ischemia rather than by a direct action of ET-1 on brain tissue.

Modification of ionotropic glutamate receptor-mediated processes in the rat hippocampus following repeated, brief seizures.

The seizure-induced molecular and functional alterations of glutamatergic transmission in the hippocampus have been investigated. Daily repeated epileptic seizures were induced for 12 days by intraperitoneal administration of 4-aminopyridine (4-AP; 4.5 mg/kg) in adult Wistar rats. The seizure symptoms were evaluated on the Racine's scale. One day after the last injection, the brains were removed for in vitro electrophysiological experiments and immunohistochemical analysis. The glutamate receptor subunits NR1, NR2A, NR2B, GluR1, GluR1(flop), GluR2, and KA-2 were studied using the histoblotting method. The semi-quantitative analysis of subunit immunoreactivities in hippocampal layers was performed with densitometry. In the hippocampus, increase of GluR1, GluR1(flop) and NR2B immunostaining was observed in most of the areas and layers. The significant decrease of GluR2 staining intensity was observed in the CA1 and dentate gyrus. Calcium permeability of hippocampal neurons was tested by a cobalt uptake assay in hippocampal slices. The uptake of cobalt increased in the CA1 area and dentate gyrus, but not in the CA3 region following 4-AP treatment. Effects of AMPA and NMDA (N-methyl-d-aspartate) glutamate receptor antagonists (1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466) and D-APV respectively) were measured in hippocampal slices using extracellular recording. Analysis of the population spikes revealed the reduced effectiveness of the AMPA receptor antagonist GYKI 52466, while the effect of the NMDA receptor antagonist d-(2R)-amino-5-phosphonovaleric acid was similar to controls. The results demonstrated that repeated convulsions induced structural and functional changes in AMPA receptor-mediated transmission, while NMDA and kainate receptor systems displayed only alterations in receptor subunit composition.

Laminar analysis of initiation and spread of epileptiform discharges in three in vitro models.

Overexcitation of neuronal networks in some forebrain structures and pathological synchronization of neuronal activity play crucial role in epileptic seizures. Seizure activity can be elicited experimentally by different convulsants. Because of various distribution of excitatory and inhibitory connections in the neocortex there might be laminar differences in seizure sensitivity. Current source density (CSD) analysis or immunocytochemical c-Fos localization offer suitable tools to localize increased activation of neurons during seizure. In the present experiments, interictal epileptiform activity elicited by 4-aminopiridine, bicuculline or Mg(2+)-free solution was recorded with a 16-channel multielectrode assembly in different layers of the somatosensory cortex, and CSDs were calculated. Parallel c-Fos immunocytochemistry was applied. Each convulsant elicited characteristic activation pattern. 4-aminopiridine induced relatively short discharges, which were associated with a huge sink in layer V, the sink and source pattern was relatively simple. Mg(2+)-free solution elicited the longest discharges, sinks appeared typically in the supragranular layers II and III than quickly distributed toward layers V and VI. Bicuculline induced rather similar seizure pattern as Mg(2+)-free solution, but the amplitudes of field potentials were larger, while the durations shorter. The peak of c-Fos activation, however, was not parallel with the largest electrical activation. Larger amount of stained cells appeared in layers II and III in 4-aminopiridine and bicuculline, respectively. In Mg(2+)-free solution the highest c-Fos activity was detected in upper layer VI. Long-lasting cellular effects do not always correspond to the largest electrical responses, which are primarily determined by the activation of asymmetrical pyramidal neurons. Interneurons, which possess more symmetric process arborisation, play less important role in the generation of field potentials, although they may be intensively activated during seizure.

Enhancement of synaptic strength in the somatosensory cortex following nerve injury does not parallel behavioural alterations.

Following infraorbital nerve transection, underlying mechanisms of the altered synaptic strength were studied in rat barrel cortex slice experiments. In addition to the in vitro electrophysiological studies, open-field tests were run to detect possible behavioural changes associated with cortical oversensitization. Enhanced NMDA receptor-mediated component of the evoked field response appeared in the barrel cortex after nerve injury. The alteration was transient, very distinct on the first day following injury, and almost returned to normal level by the end of the second week. Behavioural changes had not followed this time-course since long-lasting alterations were detected in the open-field test. These observations are in agreement with findings that showed biphasic regenerative processes following nerve injuries in other cortical areas.

Long-term changes of activity of cortical neurons after status epilepticus induced at early developmental stages in rats.

Spontaneous activity of cortical neurons was studied under urethane anesthesia in adult rats 3 months after convulsive status epilepticus induced by lithium-pilocarpine administration at the age of 12 (SE12 group) or 25 (SE25 group) days. Whereas random firing neurons dominated in control animals (61 out of 98 cells), SE25 animals exhibited a significant increase in the incidence of bursting cells (38 out of 59 units). Similar change in SE12 animals did not reach the level of statistical significance. Status epilepticus at an early developmental stage may result in a long-lasting change in functions of surviving cortical neurons.

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.

Optical recording of spreading depression in rat neocortical slices.

A spreading depression (SD) was elicited in adult rat neocortical slices by microdrop application of high potassium and the SD propagation pattern was analyzed by recording simultaneously the extracellular DC potential and the changes in the intrinsic optical signal. The electrical SD with an average peak amplitude of 13.2+/-3.4 mV showed a good spatial and temporal correlation with the optical signal. In 79% of the slices, the SD was characterized by an initial increase of light reflectance by 2.3+/-1.6%, followed by a reflectance decrease of 0.5+/-2.4% and finally a larger and long-lasting increase by 5+/-2.4%. In the remaining slices, the SD revealed an initial decrease in light reflectance by 5.8+/-1.8% followed by an increase of 1.4+/-1.2%. In all slices, the recovery in the DC recording was faster as in the optical signal. The SD preferentially propagated within layers I-IV and could be blocked in most experiments by a vertical incision through upper layers or by local glutamate receptor blockade following microdrop application of kynurenic acid in layers II-III. The SD could be also blocked by bath application of kynurenic acid, MK-801 and octanol, but not by the more specific gap junction blocker carbenoxolone. Our results indicate that the high density of dendritic processes and glutamate receptors in layers II-IV promote the horizontal spread of the SD in these cortical layers and that gap junctions are not required for the propagation of SD in neocortical slices.

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.

Anticonvulsive effect of AMPA receptor antagonist GYKI 52466 on 4-aminopyridine-induced cortical ictal activity in rat.

The effect of GYKI-52466 (1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine), a selective antagonist of AMPA receptor was investigated on the generation and manifestation of 4-aminopyridine-induced cortical epileptiform activity. In vivo experiments were carried out on pentobarbital-anaesthetised adult rats. Ictal epileptiform activity was induced by local application of 4-aminopyridine (4-Ap) to the surface of somatosensory cortex. In one group of animals, GYKI 52466 was administered intraperitoneally before 4-Ap application, in another group, the already active primary focus was treated locally by GYKI 52466. Different parameters of epileptic activity were measured and compared in GYKI 52466-treated and control animals. The results demonstrate that GYKI 52466 exerts anticonvulsive effects on both the induction and the expression of epileptiform activity, by delaying the onset of the first ictal event, decreasing the numbers and duration of ictal periods, as well as the amplitudes of epileptiform discharges both in the primary and mirror foci. However, seizure propagation to other cortical areas seemed to be facilitated. The anticonvulsive effect of GYKI 52466 was stronger in pretreatment than in treatment of ongoing epileptiform activity. As a conclusion, it is supposed that AMPA receptors are probably more dominant in the induction of epileptiform activity than in the maintenance of it, mainly through the activation of corticothalamo-cortical networks. It is also supposed that the cortical inhibition which blocks the propagation of epileptiform process might be activated mainly through non-N-methyl-D-aspartate receptors.

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.

Non-NMDA receptor antagonist GYKI 52466 suppresses cortical afterdischarges in immature rats.

GYKI 52466 (1-(4-aminophenyl)-4-methyl-7,8-methylendioxy-5H-2,3-benzo-diaz epi ne), a non-competitive non-NMDA receptor antagonist, was tested against epileptic afterdischarges elicited by cortical stimulation in 12-, 18- and 25-day-old rats with implanted electrodes. Shortening of afterdischarges and a decrease in intensity of clonic movements accompanying both stimulation and afterdischarges were induced by the 20 mg/kg dose of GYKI 52466 in 18- and 25-day-old animals, whereas 12-day-old rat pups exhibited only shortening of electroencephalographic afterdischarges. The 10 mg/kg dose of GYKI 52466 did not significantly change afterdischarges in any age group. Motor skills were compromised after the 20 mg/kg dose of GYKI 52466. This effect was again more marked in 18- and 25-day-old animals than in the youngest group. In addition, anxiolytic-like action was observed in the jumping down test in 25-day-old rats. This effect was not influenced by a benzodiazepine antagonist flumazenil. On the contrary, the anticonvulsant action of GYKI 52466 was partly blocked by flumazenil, indicating thus multiple mechanisms of action of GYKI 52466.

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.