Induced Epileptiform Activity Research Articles

Region- and pattern-specific effects of glutamate uptake blockers on epileptiform activity in rat brain slices

Many epileptic syndromes develop into pharmaco-resistant forms, calling for the development of new anticonvulsant strategies. The transmitter glutamate serves a double role as excitatory transmitter and as precursor for GABA, thus interfering with glutamate uptake may therefore exert complex effects on excitation-inhibition-balance in epileptic networks. In the present study we tested the effect of two different glutamate uptake blockers on acutely induced epileptiform activity in hippocampal-entorhinal cortex slices from adult rats: dihydrokainate (DHK) which blocks predominantly glial glutamate uptake, and threo-beta-benzyloxyaspartic acid (TBOA) which blocks both glial and neuronal glutamate uptake. Three different models were used to induce epileptiform discharges: (i) increasing NMDA receptor-mediated excitation by omitting Mg(2+)-ions; (ii) blocking potassium channels by 4-aminopyridine; (iii) reducing GABA(A) receptor-mediated inhibition by penicillin. Application of DHK or TBOA markedly reduced the frequency of epileptiform discharges in CA1 in the low magnesium and the 4-AP model while pathological activity was increased in the penicillin-model. In contrast, frequency of epileptiform discharges in EC was consistently increased by DHK and TBOA. Effects of DHK were more easily reversible than those of TBOA. Thus glutamate uptake blockers exert variable effects on epileptiform activity, depending on brain region and on the mechanism of ictogenesis.

Glutamate uptake shapes low-[Mg 2+] induced epileptiform activity in juvenile rat hippocampal slices

A wide range of data support a role for ambient glutamate (Glu) in epilepsy, although temporal patterns associated with the cellular uptake of Glu have not been addressed in detail. We report on the effects of Glu uptake inhibitors on recurrent seizure-like events (SLEs) evoked by low-[Mg 2+] condition in juvenile rat hippocampal slices. Effects were compared for inhibitors such as l-trans-pyrrolidine-2,4-dicarboxylate ( tPDC), dl-threo-β-benzyloxyaspartate ( dl-TBOA) and dihydrokainic acid (DHK), representing different transporter specificity and transportability profiles. Latency to the first SLE after drug application was shortened by the inhibitors (in % of control: 500 μM tPDC: 54 ± 7, 15 μM dl-TBOA: 74 ± 5, 50 μM dl-TBOA: 70 ± 6, 100 μM DHK: 69 ± 4, 300 μM DHK: 71 ± 7). Further SLEs were frequently aborted by higher inhibitor concentrations applied (500 μM tPDC: 2/6, 50 μM TBOA: 5/5, 100 μM DHK: 6/8, 300 μM DHK: 3/3). Simultaneous field potential and whole-cell voltage recordings showed depolarization-induced inactivation of CA3 pyramidal neurons during inhibitor application. In the presence of inhibitors, the amplitude of forthcoming SLE was also decreased (in % of control: 500 μM tPDC: 66 ± 9, 15 μM dl-TBOA: 88 ± 5, 50 μM dl-TBOA: 59 ± 6, 100 μM DHK: 67 ± 4, 300 μM DHK: 68 ± 1). Dependent on type and concentration of the inhibitor, the duration of the first SLE of drug application either increased (100 μM DHK: 375 ± 90 %; 100 μM tPDC: 137 ± 13 %) or decreased (50 μM TBOA: 62 ± 13 %; 300 μM DHK: 60 ± 15 %) reflecting differences in subtype-specificity or mechanism of action of the inhibitors. Our findings suggest a role for ambient Glu in the genesis and maintenance of recurrent epileptiform discharges.

BDNF-TrkB signaling pathway mediates the induction of epileptiform activity induced by a convulsant drug cyclothiazide

Brain-derived neurotrophic factor (BDNF) and its receptor TrkB play an important function in neuronal development and synaptic plasticity. Recently we have established that cyclothiazide (CTZ) is a novel convulsant drug inducing robust epileptiform activity in hippocampal neurons both in vitro and in vivo. However, the molecular mechanisms underlying such convulsant action of CTZ are unknown. Here, we investigated potential roles of BDNF-TrkB signaling pathway in the CTZ-induction of epileptiform activity. In anaesthetized rats, CTZ dose-dependently induced epileptiform activity characterized by progressing of multiple peaks of population spikes, spontaneous spiking events, and synchronized epileptiform bursts. Pre-injection of a receptor tyrosine kinase inhibitor K252a or a specific antibody for TrkB receptors before intracerebroventricular injection of CTZ significantly suppressed the epileptiform activity induced by CTZ. Similarly, in cultured hippocampal pyramidal neurons, pre-treatment with CTZ together with K252a or TrkB-receptor antibody also inhibited the CTZ-induction of epileptiform activity. Furthermore, we demonstrated that acute application of K252a in hippocampal cultures inhibited epileptiform bursts and action potential firing. We conclude that activation of BDNF-TrkB signaling pathway is fundamentally important during the CTZ-induction of epileptiform activity both in vitro and in vivo.

Activity-dependent induction and maintenance of epileptiform activity produced by group I metabotropic glutamate receptors in the rat hippocampal slice

Activation of group I metabotropic glutamate receptors (mGluRs) produces a long-lasting change in hippocampal excitability that persists in the absence of an agonist. Exposure to the group I mGluR agonist dihydroxyphenylglycine (DHPG) results in the induction of spontaneously occurring epileptiform activity in the CA3 region of rat hippocampal slices that includes both brief interictal discharges and longer synchronous activity that resembles seizure or ictal activity (>2s duration oscillating at a frequency greater than 2 Hz). We evaluated activity-dependent mechanisms for the induction and maintenance of epileptiform activity. Both the induction and maintenance of epileptiform activity was blocked by inhibiting action potential generation with tetrodotoxin or substitution of sodium with choline or by blocking AMPA/KA ionotropic glutamate receptors. The ictal epileptiform activity induced by DHPG was composed of synchronous synaptic activity. Antagonists of group I mGluRs, either mGluR1 or mGluR5, suppressed the induction of ictal activity but had minimal effects on the maintenance of epileptiform activity. Group I mGluRs activate phospholipase C and inhibition of phospholipase C suppressed the induction but not the maintenance of epileptiform activity. Taken together, these results point to a use dependent change in CA3 neuronal network function produced by group I mGluR activation. Furthermore, activation of both mGluR1 and 5 is required to induce ictal discharges. The induction of epileptiform activity by DHPG is an in vitro model of epileptogenesis, and the development of epileptiform activity in this model depends on neuronal activity and synaptic transmission.

Effects of glucose and glutamine concentration in the formulation of the artificial cerebrospinal fluid (ACSF)

The composition of the ACSF is fundamental in controlling the extracellular environment of the brain slice preparation. ‘Typical’ formulations lack amino acids and contain a higher concentration of glucose (10 mM) than in the cerebrospinal fluid (0.47 – 4.4 mM). We examined the effects of different concentrations of glutamine, the most abundant amino acid in the CSF, and glucose on rat hippocampal slice physiology. Bipolar paired-pulse stimulation was applied to the Schaffer collaterals and population spikes were monitored in the CA1 pyramidal layer for ~1 hour. Addition of glutamine (0.5 mM) to slices superfused with 10 mM of glucose did not enhance population spike amplitude. Higher concentration of glutamine (2-5 mM) resulted in spreading-depression. Decreasing glucose concentration from 10 mM to 5 mM, in the absence of glutamine, attenuated population spikes. Decreasing glucose to 2 mM, in the absence of glutamine, suppressed evoked population spikes. Superfusing brain slices with ACSF containing ‘physiological’ concentrations of both glucose (2 mM) and glutamine (0.5 mM) similarly suppressed population spikes. In separate experiments, during high-K + induced epileptiform activity, glutamine (0.5 mM) did not affect the burst duration, frequency or waveform. These results suggest that the concentration of glucose in ACSF should conservatively be 10 mM in order to maximize paired-pulse population responses while the presence of physiological concentration of glutamine (0.5 mM) has minimal effects on paired-pulse responses and high-K + induced epileptiform activity. These results are discussed in the context of fundamental differences between in vitro brain slice superfusion and in vivo brain perfusion.

Antiepileptic effects of electroacupuncture vs vagus nerve stimulation on cortical epileptiform activities

Introduced about two decades ago, vagus nerve stimulation (VNS) therapy has been increasingly used for the treatment of refractory epilepsy recently. This study was set out to compare the effects between VNS and electroacupuncture (EA) on pentylenetetrazole (PTZ) induced epileptiform activities in the rat cerebral cortex. Under general anesthesia, the parietal cortex of the rat (n=20) was exposed to record the cortical epileptiform activities. The left vagus nerve was stimulated at 30 Hz, 1 mA or 3 mA for 5 min. For EA, "Dazhui" acupoint (GV14) was stimulated with a pair of acupuncture needles with the same parameters. The results show that both VNS and EA at either 1 mA or 3 mA could inhibit the PTZ-induced cortical epileptiform activities, and higher stimulation (3 mA) was not associated with a greater inhibition. In the cases that showed inhibitory responses, there were no statistically significant differences between the two modalities, implying that EA could be comparable to VNS in the treatment of epilepsy. Thus, under current experimental settings, the antiepileptic effect induced by electrical stimulation appeared not vagal specific, and EA could be a good alternative to VNS in the management of epilepsy.

Antiepileptic activity of zonisamide on hippocampal CA3 neurons does not depend on carbonic anhydrase inhibition

Zonisamide (ZNS) is an anticonvulsant drug known to affect various neuronal channels and transmitter systems. ZNS has also been reported to inhibit carbonic anhydrase activity and may thus influence neuronal activity via changes of pH. Therefore, we analyzed effects of ZNS in vitro using epileptic model systems which are sensitive to carbonic anhydrase inhibition and pH changes. Intracellular recordings from CA3 neurons (hippocampal slice, adult guinea pigs) were carried out under bicarbonate-buffered conditions. Epileptiform activity was induced by either 4-aminopyridine or theophylline. In parallel experiments, intracellular pH (pHi) was determined in the CA1 and CA3 subfields of 2',7-bis(2-carboxyethyl)-5(6)-carboxyfluorescin-acetoxymethyl ether (BCECF-AM) loaded slices. The ammonium prepulse method was used to test for effects of ZNS on pHi regulation. ZNS (50 microM) reversibly reduced the frequency of 4-AP induced epileptiform bursting and the number of action potentials per bursts but had no effect on input resistance and membrane potential. Theophylline-induced epileptiform bursting, although sensitive to hypercapnic acidosis, was not affected by ZNS. There was also no effect on steady-state pHi and pHi regulation of BCECF-AM loaded hippocampal tissue. Clinically relevant concentrations of ZNS strongly inhibit 4-AP induced epileptiform activity of hippocampal CA3 neurons in vitro, but this effect was unlikely based on carbonic anhydrase inhibition or changes of neuronal pHi.

Neuronal Desynchronization as a Trigger for Seizure Generation

Experimental reports have appeared which challenge the dogma that epileptic seizures arise as a consequence of neuronal hypersynchronization. We sought to explore what mechanisms that desynchronize neuronal firing could induce epileptic seizures. A computer model of connections in a mammalian hippocampal slice preparation was constructed including two recently-reported distinct inhibitory feedback circuits. When inhibition by interneurons that synapse on pyramidal dendrites was decreased, highly localized seizure-like bursting was observed in the CA3 region similar to that which occurs experimentally under GABAergic blockade. In contrast, when inhibition by interneurons that synapse in the axosomatic region was similarly decreased, no such bursting was observed. However, when this transient inhibition was increased, normal coordinated spread of excitation was interrupted by high-frequency localized seizure-like bursting. The increase of this inhibitory input resulted in decreased cell coupling of pyramidal neurons. A decrease in phase coherence was initially observed until seizure-like activity initiated causing a net increase in coherence as has been observed in epileptic patients. These results provide a possible pathway in which a decrease in synchronization could provide the trigger for inducing epileptiform activity.

Repeated blockade of GABAA receptors in the medial septal region induces epileptiform activity in the hippocampus

Changes in the activity of putative interneurons of the stratum oriens of the hippocampus and hippocampal EEG after the delivery of the GABAA receptor antagonist bicuculline (1.0nmol/1microl) to the medial septal region were investigated in awake rabbits. The injection of bicuculline produced a sharp increase in the firing rate in 94.3% of hippocampal cells. The effect of bicuculline on the neuronal theta rhythmicity depended on the number of injections. The first five daily infusions decreased the theta activity in 85.7% of cells. On the fourth to fifth experimental days paroxysmal discharges and 8-15Hz oscillations were recorded in the hippocampal EEG. Six to seven further daily bicuculline injections following a brief diminution of theta activity produced a sharp augmentation of theta oscillations in 78.9% of cells and provoked seizures. Immediately before seizures, stabilization of theta bursts and an increase in burst frequency was usually observed in putative interneurons. During seizures, neuronal rhythmic activity was either disordered and then turned into seizure discharges or was inhibited, partially or completely. In the hippocampal EEG, the power of theta rhythm before seizures usually strongly increased compared with controls. Injection of the GABAA agonist muscimol (30nmol/1microl) 15min before bicuculline infusion prevented the development of seizures. These findings suggest that the interplay between septal neurons via GABAA receptors is critical in the tuning of septal output signals that insure generation of natural theta rhythm as well as adequate functioning of the hippocampus.

K+-Channel Openers Suppress Epileptiform Activities Induced by 4-Aminopyridine in Cultured Rat Hippocampal Neurons

K+ channels are key modulators of neuronal excitability, and mutations in certain types of these channels are known to cause epileptic seizures. Activation of K+ channels is reported to suppress epileptic discharge; however, the types of K+-channel openers that are most effective as anti-epileptic agents are not well understood. We established a quantitative fluorescence assay using the Na+ indicator sodium-binding benzofuran isophthalate (SBFI) for evaluation of various compounds on epileptiform activities induced by 4-aminopyridine (4-AP) in cultured rat hippocampal neurons. Among the K+-channel openers, the KV7.2/KV7.3-channel openers retigabine and flupirtine and KCa2-channel openers NS309, DCEBIO, and 1-EBIO showed potent anti-epileptic effects similar to conventional antiepileptic drugs (AEDs). In contrast, the KCa1.1-channel openers NS1619, isopimaric acid, and chlorzoxazone demonstrated moderate inhibition. The Kir6-channel openers minoxidil, cromakalim, and pinacidil did not show anti-epileptic effects. We concluded that KV7.2/KV7.3, KCa2, and, to some extent, KCa1.1-channel openers, but not Kir6-channel openers, suppress 4-AP–induced epileptiform activities in hippocampal neurons. These results suggest that the K+-channel openers for this category of K+channels might have therapeutic potential as new classes of antiepileptic drugs.

Acute induction of epileptiform discharges by pilocarpine in the in vitro isolated guinea-pig brain requires enhancement of blood–brain barrier permeability

Systemic application of the muscarinic agonist, pilocarpine, is commonly utilized to induce an acute status epilepticus that evolves into a chronic epileptic condition characterized by spontaneous seizures. Recent findings suggest that the status epilepticus induced by pilocarpine may be triggered by changes in the blood–brain barrier (BBB) permeability. We tested the role of the BBB in an acute pilocarpine model by using the in vitro model brain preparation and compared our finding with in vivo data. Arterial perfusion of the in vitro isolated guinea-pig brain with <1 mM pilocarpine did not cause epileptiform activity, but rather reduced synaptic transmission and induced steady fast (20–25 Hz) oscillatory activity in limbic cortices. These effects were reversibly blocked by co-perfusion of the muscarinic antagonist atropine sulfate (5 μM). Brain pilocarpine measurements in vivo and in vitro suggested modest BBB penetration. Pilocarpine induced epileptiform discharges only when perfused with compounds that enhance BBB permeability, such as bradykinin (n=2) or histamine (n=10). This pro-epileptic effect was abolished when the BBB-impermeable muscarinic antagonist atropine methyl bromide (5 μM) was co-perfused with histamine and pilocarpine. In the absence of BBB permeability enhancing drugs, pilocarpine induced epileptiform activity only after arterial perfusion at concentrations >10 mM. Ictal discharges correlated with a high intracerebral pilocarpine concentration measured by high pressure liquid chromatography.We propose that acute epileptiform discharges induced by pilocarpine treatment in the in vitro isolated brain preparation are mediated by a dose-dependent, atropine-sensitive muscarinic effect promoted by an increase in BBB permeability. Pilocarpine accumulation secondary to BBB permeability changes may contribute to in vivo ictogenesis in the pilocarpine epilepsy model.

Enhanced Astrocytic Ca2+Signals Contribute to Neuronal Excitotoxicity after Status Epilepticus

Status epilepticus (SE), an unremitting seizure, is known to cause a variety of traumatic responses including delayed neuronal death and later cognitive decline. Although excitotoxicity has been implicated in this delayed process, the cellular mechanisms are unclear. Because our previous brain slice studies have shown that chemically induced epileptiform activity can lead to elevated astrocytic Ca2+ signaling and because these signals are able to induce the release of the excitotoxic transmitter glutamate from these glia, we asked whether astrocytes are activated during status epilepticus and whether they contribute to delayed neuronal death in vivo. Using two-photon microscopy in vivo, we show that status epilepticus enhances astrocytic Ca2+ signals for 3 d and that the period of elevated glial Ca2+ signaling is correlated with the period of delayed neuronal death. To ask whether astrocytes contribute to delayed neuronal death, we first administered antagonists which inhibit gliotransmission: MPEP [2-methyl-6-(phenylethynyl)pyridine], a metabotropic glutamate receptor 5 antagonist that blocks astrocytic Ca2+ signals in vivo, and ifenprodil, an NMDA receptor antagonist that reduces the actions of glial-derived glutamate. Administration of these antagonists after SE provided significant neuronal protection raising the potential for a glial contribution to neuronal death. To test this glial hypothesis directly, we loaded Ca2+ chelators selectively into astrocytes after status epilepticus. We demonstrate that the selective attenuation of glial Ca2+ signals leads to neuronal protection. These observations support neurotoxic roles for astrocytic gliotransmission in pathological conditions and identify this process as a novel therapeutic target.

Neurons Skip a Beat during Fast Ripples

Fast ripples are EEG transients emanating from epileptic foci, but fast-ripple frequencies far exceed maximal neuronal firing rates. In this issue of Neuron, Menendez de la Prida and coworkers propose that out-of-phase firing of a subpopulation of neurons during physiological ripple activity effectively doubles the ripple frequency to produce fast ripples.

The role of nitric oxide in the anticonvulsant effects of pyridoxine on penicillin-induced epileptiform activity in rats

The present study was conducted to identify the role of nitric oxide (NO) in the anticonvulsant effects of pyridoxine hydrochloride on penicillin-induced epileptiform activity in rats. A single microinjection of penicillin (500 units) into the left sensorimotor cortex induced epileptiform activity within 2-4 min, progressing to full seizure activity lasting about 3-5h. Thirty minutes after penicillin injection, 20, 40, 80, and 160 mg/kg of pyridoxine hydrochloride was administered intraperitoneally (i.p.). Pyridoxine significantly reduced the frequency of penicillin-induced epileptiform activity. A low dose of pyridoxine (40 mg/kg) was the most effective in reducing both the frequency and amplitude of epileptiform activity. The effect of systemic administration of nitric oxide synthase (NOS) inhibitors, non-selective N(G)-nitro-L-arginine methyl ester (L-NAME), selective neuronal NOS inhibitor, 7-nitroindazole (7-NI) and NO substrate, L-arginine on anticonvulsive effects of pyridoxine was investigated. The administration of L-arginine (500 mg/kg, i.p.) and 7-NI (25 and 50 mg/kg, i.p.) significantly decreased the frequency of epileptiform electrocorticographical (ECoG) activity while administration of L-NAME (60 mg/kg, i.p.) and the inactive form of arginine (D-arginine) did not influence it. The administration of L-NAME (60 mg/kg, i.p.) 15 min before pyridoxine (40 mg/kg i.p.) application reversed the anticonvulsant effects of pyridoxine whereas 7-NI (25 and 50 mg/kg, i.p.) did not influence it. The same dose of its inactive enantiomer N(G)-nitro-D-arginine methyl ester (d-NAME) failed to reverse the anticonvulsant effects of pyridoxine. The administration of L-arginine (500 mg/kg, i.p.) did not affect the frequency of epileptiform ECoG activity in the pyridoxine administered group. L-arginine did not reverse the anticonvulsant effect of 7-NI in the penicillin and pyridoxine administered groups. The results of present study indicate that the inhibitory effect on the anticonvulsant activity of pyridoxine against penicillin-induced epileptiform activity was produced by L-NAME, not by 7-NI, and is probably not related to the decrease of NOS activity in the brain.

Forskolin potentiates the paraoxon-induced hyperexcitability in snail neurons by blocking afterhyperpolarization

One characteristic of organophosphate poisoning is the ability to increase excitability or induce epileptiform activity in nerve cells, but underlying mechanisms are not fully understood. We have previously reported that paraoxon, an organophosphate compound, at submicromolar concentrations effectively suppress Ca 2+ spikes and modulate the activity of snail neurons. This effect was unrelated to acetylcholinesterase (AChE) inhibition but was found to involve the direct or indirect modulation of ion channels [Vatanparast J, Janahmadi M, Asgari AR, Sepehri H, Haeri-Rohani A. Paraoxon suppresses Ca 2+ spike and afterhyperpolarization in snail neurons: relevance to the hyperexcitability induction. Brain Res 2006a;1083(1):110–7]. In the present study, the interaction of paraoxon with cAMP formation on the modulation of Ca 2+ spikes and neuronal excitability was examined. Forskolin, the activators of adenylate cyclase, suppressed afterhyperpolarization (AHP) and increased the activity of snail neurons without any significant effect on the Ca 2+ spike duration. Pretreatment with forskolin, although attenuated the suppressing effect of paraoxon on the duration of Ca 2+ spikes but also potentiated the paraoxon-induced hyperexcitability by enhancing the suppressive effects of paraoxon on AHP. Our findings support the possible involvement of cAMP formation in the paraoxon-induced AHP suppression and neuronal hyperexcitability, although activation of cAMP pathway may attenuates some effects of paraoxon.

Anticonvulsant effects of damage to structures involved in seizure induction in rats exposed to soman

In nerve agent research, it is assumed that the regions from which seizure activity is triggered may offer clues for the designing of effective anticonvulsive therapy. In the present study, selective brain lesions were made to identify critical cholinergic pathways and seizure controlling areas involved in the induction of epileptiform activity in rats challenged with soman. The results showed that rats with bilateral aspiration lesion of the seizure controlling substrate, area tempestas (AT) in the piriform cortex, displayed marked anticonvulsant effects, whereas such effects were not seen when substantia nigra was destroyed. Aspiration lesion of the medial septal area (MS) including the vertical limb of the diagonal band nucleus (DBN) caused increased latency to the onset of convulsions, whereas damage to the nucleus basalis magnocellularis (NBM), nucleus accumbens, or both MS and NBM did not cause anticonvulsant effects. Saporin lesion of MS, DBN (horizontal limb), or MS + DBN had no anticonvulsant effects, suggesting that aspiration lesion of MS disrupted pathways beyond cholinergic ones. Severe aphagia/adipsia and reduced body weight occurred in rats with lesions in the septal area. In separate sham operated rats, a strong positive correlation was found between body weight and latency to onset of convulsions in response to soman. Thus, weight loss and a relatively high dose of soman (1.6 × LD 50) in this context may have masked potential anticonvulsant effects among some lesioned animals. It is inferred that MS and AT/piriform cortex occur as prime target areas for induction of seizures by soman.

The Effects of Ascorbic Acid on Penicillin‐induced Epileptiform Activity in Rats

Epileptic seizure results from excessive discharge in a population of hyperexcitable neurons. A number of studies help to document the effects of active oxygen free radical scavengers such as alpha-tocopherol or ascorbic acid (vitamin C). In the present study, we examined the effects of ascorbic acid, at the six different doses, on penicillin-induced epileptiform activity. A single microinjection of penicillin (2.5 microl, 500 units, intracortically) into the left sensorimotor cortex induced epileptiform activity within 2-5 min, progressing to full seizure activity lasting approximately 3-5 h. In the first set of experiments, 30 min after penicillin injection, six different doses of ascorbic acid (25, 50, 100, 200, 400, or 800 mg/kg) were administered intraperitoneally (IP). The other group of animals received the effective dose of ascorbic acid (100 mg/kg, IP) for 7 days. Ascorbic acid administration was stopped 24 h before penicillin treatment. Another group of rats received the effective dose of ascorbic acid (100 mg/kg, IP) 30 min before penicillin treatment. In the second set of experiments, the lipid peroxidation (MDA) and reduced glutathione (GSH) levels of brain were measured in the control, control + ascorbic acid, penicillin, and penicillin + ascorbic acid groups. Ascorbic acid, at the low dose (50, 100 mg/kg, 30 min after penicillin injection), decreased both the frequency and amplitude of penicillin-induced epileptiform activity in rats. Ascorbic acid, at intermediate doses (200, 400 mg/kg, 30 min after penicillin injection), decreased the frequency of epileptiform activity without changing the amplitude. Ascorbic acid, at the lowest dose (25 mg/kg) and highest dose (800 mg/kg) (30 min after penicillin injection), did not change either the frequency or amplitude of epileptiform activity. Ascorbic acid, at the low dose (100 mg/kg) was the most effective dose in changing the frequency and amplitude of penicillin-induced epileptiform activity. Pretreatment with ascorbic acid (100 mg/kg) 30 min before penicillin treatment caused a significant delay in the onset of penicillin-induced epileptiform activity. Pretreatment with ascorbic acid (100 mg/kg) for 7 days did not change the latency of epileptiform activity. The most effective dose of ascorbic acid (100 mg/kg) prevented both the decrease in GSH level and the increase in lipid peroxidation level (MDA) occurring after penicillin-induced epileptiform activity. These data indicate that ascorbic acid has neuroprotective activity against penicillin-induced epileptiform electrocorticogram activity.

Expression of Adhesion Factors Induced by Epileptiform Activity in the Endothelium of the Isolated Guinea Pig Brain In Vitro

Brain inflammation has been recently considered in the pathogenesis of focal epilepsies. Synthesis of pro-inflammatory mediators in the brain was described both in experimental models of seizures and in human postsurgical tissue. Inflammatory mediators may up-regulate endothelial adhesion molecules, therefore promoting adhesion and homing of leucocytes into the brain. In the present study, expression of inducible adhesion factors in brain endothelium was verified after pharmacological induction of seizure-like activity in specific brain areas of the in vitro isolated guinea pig brain. Experiments were performed in isolated guinea-pig brains maintained in vitro by arterial perfusion. In this preparation, brief application of the GABAa receptor-antagonist, bicuculline, consistently induced focal ictal discharges in the limbic region that secondarily diffuse to the neocortex, as verified by simultaneous electrophysiological recording of extracellular activity. At the end of the electrophysiological experiment (after 5 h in vitro), brains were fixed and immunostaining for adhesion molecules P-selectin and ICAM-1 and for Fos protein was evaluated. Immunohistochemical analysis of isolated brains in which seizure-like activity was induced revealed expression of inducible adhesion factors P-selectin and ICAM-1 in the endothelium of small-medium size brain vessels. In particular, the expression of these molecules was consistently observed in all areas involved in epileptic seizure-like ictal activity (limbic cortices and neocortex), and was infrequently found in regions that generated interictal spiking (piriform cortex), suggesting a trigger role played by seizures for endothelial activation. An increase in Fos protein expression was evident in all analyzed limbic areas and in the neocortex, indicating a correlation between the areas of neuronal and endothelial activation. In control brains maintained in vitro for comparable times without induction of epileptiform activity, no immunoreactivity for Fos and adhesion molecules was observed. Seizure-like activity in an in vitro isolated brain preparation induces the expression of adhesion molecules in the cerebral endothelium. These observations indicate that local endothelial activation may represent a crucial step for the development of an inflammatory response induced by seizures, and suggest a possible novel pathogenic mechanism during the process of epileptogenesis.

The influence of ethanol intake and its withdrawal on the anticonvulsant effect of α-tocopherol in the penicillin-induced epileptiform activity in rats

Previous studies proposed the existence of a relationship between epilepsy and ethanol. Ethanol may have either proconvulsive or anticonvulsive effects on epileptic activity in different experimental epilepsy models. The influence of high dose ethanol intake and its withdrawal on the anticonvulsant effect of α-tocopherol was examined after intracortical injection of penicillin (500 units) to induce epileptiform activity. Thirty minutes after penicillin injection, the most effective dose of α-tocopherol (500 mg/kg) was administrated intramuscularly (i.m.). Ethanol-treated rats received a daily dose of 9.0 g/kg of 30% ethanol solution via an oesophageal probe for 15 days. All rats in the withdrawal group were anesthetized for induction of penicillin-induced epileptiform activity 28 h after the last ethanol administration. The epileptiform activity was verified by electrocorticographic (ECoG) recordings. Ethanol, in a dose of 9 g/kg, significantly decreased the mean frequency of penicillin-induced epileptiform ECoG activity without changing the amplitude. The mean frequency of ECoG activity was decreased in the 60 and 70 min period from penicillin injection in the ethanol-treated + α-tocopherol and ethanol withdrawal + α-tocopherol groups compared with the penicillin-injected (500 units, i.c.) group, respectively. α-Tocopherol was more effective in decreasing the mean frequency of epileptiform activity in the ethanol + α-tocopherol group than in other α-tocopherol administrated groups. Ethanol withdrawal caused an increase in frequency of epileptiform activity in the withdrawal + α-tocopherol group compared with other α-tocopherol administrated groups. α-Tocopherol did not affect the amplitude of epileptiform activity in any group. Possible mechanisms of ethanol influence on the neuroprotective actions of α-tocopherol are still a crucial issue associated with epilepsy.

The effects of octanol on penicillin induced epileptiform activity in rats: An in vivo study

The common features of all types of epilepsy are the synchronized and uncontrolled discharges of nerve cell assemblies. The reason for the pathologically synchronized discharges of the neuron is not exactly known yet. Recent reports claim that gap junctions have a critical role in neuronal synchronization. The present study was planned to investigate the effects of octanol, a gap junction blocker, on penicillin-induced experimental epilepsy. Permanent screw electrodes allowing EEG monitoring from conscious animals and permanent cannula providing the administration of the substances to the brain ventricle were placed into the cranium of rats under general anesthesia. After the postoperative recovery period, epileptiform activity was generated by injecting 300 IU crystallized penicillin through the ventricular cannula. When epileptiform activity, monitored from a digital recording system, reached at its maximum intensity, octanol was applied in the same way as penicillin administered. Application of octanol caused an inhibition in the epileptiform activity. Vehicle solution alone did not affect the epileptiform activity. Results of this study suggest that the blockade of electrical synapses may contribute to the prevention and amelioration of epileptic activity. Production of gap junction blockers selective for connexin types is needed. Further studies on the differential roles of gap junctions on certain epileptiform activities are required.