Gamma-aminobutyric acid A receptor gamma 2 subunit following Mg-free-induced seizures in cultured developing neurons

BACKGROUND:Studies have demonstrated that in vitro cultured cortical neurons from embryonic rats can produce spontaneous recurrent epileptiform discharges following transient Mg2+-free extracellular solution culture. OBJECTIVE: To explore gamma-aminobutyric acid A receptor (GABAAR) γ2 subunit expression follow-ing Mg2+-free-induced seizures in cultured developing neurons. DESIGN,TIME AND SETTING: Cellular and molecular biology. The in vitro experiment was performed at the Department of Pediatrics,Second Xia...     

早期惊厥样放电对发育中大鼠皮层神经元远期MTT代谢率的影响

目的　探讨早期无镁细胞外液处理后惊厥样放电对发育中大鼠皮层神经元远期MTT代谢率的影响。方法 以原代培养的大鼠大脑皮层神经元无镁诱导的反复惊厥样放电为模型 ,根据对培养 6d皮层神经元的不同处理分为三组 :正常DMEM培养液组 (NOT)、正常细胞外液组 (CONT)和无镁细胞外液组 (MGF)。神经元分别在上述三种液体中孵育 3h ,然后换为正常DMEM培养液继续培养 ,在培养 7,12及 17d时测定皮层神经元MTT代谢率。结果 与CONT和NOT组相比 ,MGF组培养 7和 12d皮层神经元MTT代谢率分别为 6 2 .71%与 5 5 .4 2 % ,均明显降低 (P <0 .0 5 )。培养 17d的皮层神经元MTT代谢率三组中两两比较无统计学差异 (P >0 .0 5 )。 结论 发育中大鼠皮层神经元早期惊厥样放电可以导致神经元功能较长期的改变。     

难治性癫痫细胞模型中神经元损伤及神经突起的变化

目的 了解难治性癫痫细胞模型神经元损伤情况及神经突起的形态学变化.方法 培养至第10天的海马神经元用无镁液处理3 h,制备成难治性癫痫细胞模型,测定乳酸脱氢酶(lactic acid dehydrogenase,LDH)释放量了解神经元损伤情况,应用光学显微镜及扫描电镜观察神经元和神经突起的形态学变化.结果 模型组LDH释放量(U/L)在无镁液处理后3、6、24 h(分别为4.26±1.28、6.56±2.34和16.67±3.57)均较对照组明显升高(LSD法,P<0.05).随着时间的延长,模型组LDH释放量逐渐升高(F=39.316,P<0.05).经无镁液处理后24 h,光学显微镜下可见神经元胞体相互靠近,神经突起相互迁移聚集,突起连接成网格样变化,72 h时这种变化更加明显.扫描电镜下可见神经元胞膜粗糙,有小凹陷形成,神经突起交织聚集.结论 难治性癫痫细胞模型存在神经元损伤,存在神经突起的形态学变化.

Abstract：

Objective To establish the cell model of intractable epilepsy and to observe its neuronal damage and morphologic change of neurites.Methods The model was established by exposing hippocampal neurons to Mg2+ -free media for 3 hours on days 10 of culture.Expression of lactic acid dehydrogenase (LDH) in supernatant was measured as an index of neuronal damage.The morphologic change of neurons and neurites was observed by optical microscope and scanning electron microscope (SEM).Results Compared to the control group, level of LDH (U/L) was significantly increased in the model group at different time points (3 hours: 4.26 ± 1.28, 6 hours: 6.56 ±2.34 and 24 hours: 16.67 ±3.57, P <0.05).With time prolonging, release of LDH in the model group was gradually increased (F = 39.316,P <0.05).Under optical microscope, neurons of model group migrated closely to each other and neurite connections appeared to be gradually "reticulated" after Mg2+ -free media treatment for 24 hours; and the "reticulated" neurites connections become more obvious after 72 hours.Under SEM, neuronal membrane was rough and had several small depressions, neurites were interlaced in cluster.Conclusions Neuronal damage and morphologic change of neurites are verified in the cell model of intractable epilepsy.     

Free radical production correlates with cell death in an in vitro model of epilepsy

Free radical (FR) production, a major step in calcium-dependent neurodegeneration, has been linked to the generation of epileptiform activity and seizure-induced cell death. However, direct evidence of FR production in neurons during seizures has never been presented. Using hippocampal cultured slices we demonstrate that FRs are produced in CA3 but not CA1 pyramidal neurons during the rhythmic synchronous activity induced by the GABAA receptor antagonist bicuculline. The production of FRs (measured as changes in the fluorescence emission of dihydrorhodamine 123) was correlated with an increase in the baseline levels of intracellular calcium ([Ca2+]i) estimated by fluo-3 injected into individual neurons via a patch pipette. [Ca2+]i increased during spike bursting and returned to baseline levels after the burst termination in CA1, but not in CA3, pyramidal neurons where 'interburst' calcium concentrations progressively increased. Measurement of cell death, performed with propidium iodide 48 h after a 30-min exposure to bicuculline, revealed most prominent degeneration of pyramidal neurons in the CA3 pyramidal layer. The FR scavengers vitamin E and glutathione significantly reduced the seizure-induced neurodegeneration without supressing spontaneous epileptiform activity. These observations indicate that FR overproduction is related to seizure-induced neuronal death.     

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.     

Docosahexaenoic acid inhibits synaptic transmission and epileptiform activity in the rat hippocampus

Docosahexaenoic acid (DHA) has been suggested to be required for neuronal development and synaptic plasticity. However, in view of the fact that DHA facilitates NMDA responses and blocks K(+) channels, it might predispose the neurons to epileptiform bursting. By using extracellular recording of population spikes in the CA1 region of rat hippocampal slices, we tested this possibility by examining the effect of DHA on the epileptiform activity induced by bicuculline or in Mg(2+)-free medium. When stimuli were delivered to the Schaffer collateral/commissural pathway every 20 or 30 sec, DHA had no significant effect on the epileptiform activity. However, when the frequency of stimulation was increased to 0.2 Hz, DHA attenuated the amplitude of the bursting activity induced by bicuculline to 57.5+/- 10.8% and those induced by Mg(2+)-free ACSF to 65.8+/-13.9% of control. DHA reduced the slope of field excitatory postsynaptic potential (fEPSP) to 77.1+/-7.4% of baseline, without significant effect on the ratio of paired-pulse facilitation (PPF). By intracellular recording of neurons in the stratum pyramidale of rat hippocampal slices, we found that DHA markedly inhibited the repetitive firing of action potentials elicited by depolarizing current pulses but did not affect the initial action potential. Thus, DHA may attenuate epileptic activity mainly through the frequency-dependent blockade of Na(+) channels.     

Epileptiform activity induced by low extracellular magnesium in the human cortex maintained in vitro.

Extracellular field potentials and [K+]o were recorded in slices of human epileptogenic neocortex maintained in vitro during perfusion with Mg(2+)-free artificial cerebrospinal fluid (ACSF). The human neocortex was obtained during neurosurgical procedures for the relief of seizures that were resistant to medical treatment. Spontaneous epileptiform activity and episodes of spreading depression appeared within 1.5 to 2 hours of perfusion with Mg(2+)-free ACSF. The epileptiform discharges consisted of negative field potential shifts (amplitude, 0.8-10 mV) that lasted 2.5 to 80 seconds and recurred at intervals ranging between 4 and 160 seconds. Both duration and frequency of occurrence of epileptiform events were not significantly different when measured in slices obtained from spiking tissue compared with those gathered from nonspiking neocortical areas. Transient increases in [K+]o of up to 10.5 mM were associated with each epileptiform discharge; these changes were maximal and fastest in the middle neocortical layers. Spreading depression episodes were characterized by 20 to 30-mV negative shifts that lasted up to 200 seconds and were accompanied by increases in [K+]o of approximately 100 mM. Epileptiform discharges and spreading depressions did not occur during perfusion with Mg(2+)-free ACSF that contained either competitive or noncompetitive antagonists of the N-methyl-D-aspartate (NMDA) receptor subtype. In contrast, pharmacological blockade of non-NMDA receptors did not influence the epileptiform activity observed in Mg(2+)-free ACSF. These findings demonstrate that decreasing [Mg2+]o leads to the appearance of both spontaneous epileptiform discharges and spreading depression in the human epileptogenic neocortex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations of NR2B and PSD-95 expression after early-life epileptiform discharges in developing neurons

As an extreme form of abnormally synchronized activity, epilepsy may modify patterns of organization in the nervous system. It is clear that enhanced glutamatergic excitatory synaptic transmission with alterations in the expression of ionotropic glutamate receptors is a mechanism critical for seizure susceptibility and excitotoxicity. However, the exact quomodo and the roles of regulated N-methyl-D-aspartate receptor (NMDAR) composition and expression of a major postsynaptic density (PSD) scaffolding molecule, PSD-95, are as yet unclear. To study protein expression changes after epileptiform discharges in cultured immature rat cortical neurons, we divided cells into three groups which were transiently exposed to regular Neurobasal/B27 (control group), physiological solution (PS group) and magnesium-free physiological solution (MGF group) at cultured day 6. Neurons at three different culture ages (DIV7, DIV12 and DIV17) were collected for immunoblotting analysis. We found a decrease in expression of NR2B NMDAR subunit and PSD-95 (P<0.05) shortly after insult (within 24 h), which may show that brief magnesium-free media treatment of primary cultured rat cortical neurons, an in vitro model of seizure brain injury, has a major influence on the expression of NR2B subunit and PSD-95.     

Activin is a neuronal survival factor that is rapidly increased after transient cerebral ischemia and hypoxia in mice.

One approach for developing targeted stroke therapies is to identify the neuronal protective and destructive signaling pathways and gene expression that follow ischemic insult. In some neural injury models, the transforming growth factor-beta family member activin can provide neuroprotective effects in vivo and promote neuronal survival. This study tests if activin supports cortical neurons after ischemic challenge in vitro and if signals after cerebral ischemia involve activin in vivo. In a defined cell culture model that uses hydrogen peroxide (H(2)O(2))-free radical stress, activin addition maintained neuronal survival. H(2)O(2) treatment increased activin mRNA twofold in surviving cortical neurons, and inhibition of activin with neutralizing antibodies caused neuronal death. These data identify activin gene changes as a rapid response to oxidative stress, and indicate that endogenous activin acts as a protective factor for cortical neurons in vitro. Similarly, after transient focal cerebral ischemia in adult mice, activin mRNA increased at 1 and 4 h ipsilateral to the infarct but returned to control values at 24 h after reperfusion. Intracellular activated smad signals were detected in neurons adjacent to the infarct. Activin was also increased after 2 h of 11% hypoxia. Activin mRNA increased at 1 h but not 4 or 24 h after hypoxia, similar to the time course of erythropoietin and vascular endothelial growth factor induction. These findings identify activin as an early-regulated gene response to transient ischemia and hypoxia, and its function in cortical neuron survival during oxidative challenge provides a basis to test activin as a potential therapeutic in stroke injury.     

Carisbamate prevents the development and expression of spontaneous recurrent epileptiform discharges and is neuroprotective in cultured hippocampal neurons

Purpose: Although great advances have been made in the development of treatments for epilepsy, acquired epilepsy following brain injury still comprises approximately 50% of all the cases of epilepsy. Thus, development of drugs that would prevent or decrease the onset of epilepsy following brain injury represents an important area of research. Methods: Here, we investigated effects of carisbamate (RWJ 333369) on the development and expression of spontaneous recurrent epileptiform discharges (SREDs) and its neuroprotective potential in cultured hippocampal neurons. This model utilizes 3 h of low Mg²⁺ treatment to mimic status epilepticus (SE‐like) injury in vitro. Following the injury, networks of neurons manifest synchronized SREDs for their life in culture. Neuronal cultures were treated with carisbamate (200 μM) for 12 h immediately after the SE‐like injury. The drug was then removed and neurons were patch clamped 24 h following drug washout. Results: Treatment with carisbamate after neuronal injury prevented the development and expression of epileptiform discharges. In the few neurons that displayed SREDs following carisbamate treatment, there was a significant reduction in SRED frequency and duration. In contrast, phenytoin and phenobarbital, when used in place of carisbamate, did not prevent the development and expression of SREDs. Carisbamate was also effective in preventing neuronal death when administered after SE‐like injury. Conclusions: Carisbamate prevents the development and generation of epileptiform discharges and is neuroprotective when administered following SE‐like injury in vitro and may offer a novel treatment to prevent the development of epileptiform discharges following brain injuries.     

Neocortical potassium currents are enhanced by the antiepileptic drug lamotrigine

PURPOSE: We used field-potential recordings in slices of rat cerebral cortex along with whole-cell patch recordings from rat neocortical cells in culture to test the hypothesis that the antiepileptic drug (AED) lamotrigine (LTG) modulates K+-mediated, hyperpolarizing currents. METHODS: Extracellular field-potential recordings were performed in neocortical slices obtained from Wistar rats aged 25-50 days. Rat neocortical neurons in culture were subjected to the whole-cell mode of voltage clamping under experimental conditions designed to study voltage-gated K+ currents. RESULTS: In the in vitro slice preparation, LTG (100-400 microM) reduced and/or abolished epileptiform discharges induced by 4-aminopyridine (4AP, 100 microM; n = 10), at doses that were significantly higher than those required to affect epileptiform activity recorded in Mg2+-free medium (n = 8). We also discovered that in cultured cortical cells, LTG (100-500 microM; n = 13) increased a transient, 4AP-sensitive, outward current elicited by depolarizing commands in medium containing voltage-gated Ca2+ and Na+ channel antagonists. Moreover, we did not observe any change in a late, tetraethylammonium-sensitive outward current. CONCLUSIONS: Our data indicate that LTG, in addition to the well-known reduction of voltage-gated Na+ currents, potentiates 4AP-sensitive, K+-mediated hyperpolarizing conductances in cortical neurons. This mechanism of action contributes to the anticonvulsant effects exerted by LTG in experimental models of epileptiform discharge, and presumably in clinical practice.     

Human glioma cells induce hyperexcitability in cortical networks

Purpose: Patients with gliomas frequently present with seizures, but the factors associated with seizure development are still poorly understood. In this study, we assessed peritumoral synaptic network activity in a glioma animal model and tested the contribution of aberrant glutamate release from gliomas on glioma-associated epileptic network activity. Methods: In vitro brain slices were made from glioma-implanted mice. Using extracellular field recordings, we analyzed peritumoral epileptiform activity induced by Mg(2+) -free medium in slices from tumor-bearing animals and sham-operated controls. We assessed the effect of sulfasalazine (SAS), a blocker of system and glutamate release, on spontaneous and evoked activity in tumor-associated slices. Key Findings: Tumor-associated cortical networks were hyperexcitable. The onset latency of Mg(2+) -free-induced epileptiform activity was significantly shorter in tumor-bearing slices, and the incidence of Mg(2+) -free-induced ictal-like events was higher. Block of glutamate release from system decreased the response area of evoked activity and completely blocked Mg(2+) -free-induced ictal-like, but not interictal-like events. Significance: Control of seizures in patients with gliomas is an essential component of clinical management; therefore, understanding the origin of seizures is vital. This work provides evidence that peritumoral synaptic network activity is disrupted by tumor masses resulting in network excitability. We show that blocking glutamate release via system with SAS, a drug already approved by the U.S. Food and Drug Administration (FDA), can inhibit Mg(2+) -free-induced ictal-like epileptiform events similar to other chemicals used to decrease seizure activity. We, therefore, suggest that further studies should consider SAS a promising agent to aid in the treatment of seizures associated with gliomas.     

In vitro status epilepticus but not spontaneous recurrent seizures cause cell death in cultured hippocampal neurons

It is established that the majority but not all of the seizure-induced cell death is associated with status epilepticus while spontaneous recurrent seizures associated with epilepsy do not cause neuronal death. Extracellular effects and compensatory changes in brain physiology complicate assessment of neuronal death in vivo as the result of seizures. In this study we utilized a well-characterized in vitro hippocampal neuronal culture model of both continuous high-frequency epileptiform discharges (status epilepticus) and spontaneous recurrent epileptiform discharges (acquired epilepsy) to investigate the direct effects of continuous and episodic electrographic epileptiform discharges on cell death in a carefully controlled extracellular environment. The results from this study indicate that continuous high-frequency epileptiform discharges can cause neuronal death in a time-dependent manner. Episodic epileptiform seizure activity occurring for the life of the neurons in culture was not associated with increased neuronal cell death. Our data confirm observations from clinical and some animal studies that spontaneous recurrent seizures do not initiate cell death. The hippocampal neuronal culture model provides a powerful in vitro tool for carefully evaluating the effects of seizure activity alone on neuronal viability in the absence of various confounding factors and may provide new insights into the development of novel therapeutic agents to prevent neuronal injury during status epilepticus.     

低镁诱发急性海马切片高兴奋性的多电极记录

目的 多电极记录技术在脑片研究的应用已经远远超过了10年,然而该技术却没有广泛地用于癫痫领域的研究。用经典的致痫剂低镁人工脑脊液灌流大鼠急性海马切片,诱导产生癫痫样电活动并用多电极记录技术对其放电特征及内部传导方式进行分析。 方法 用多电极阵列持续记录灌流低镁人工脑脊液后海马各区域的放电情况,并比较切断CA3与CA1区域间的Schaffer氏纤维后各区的放电情况。 结果 在急性海马切片上诱导出自发、同步、癫痫样电活动;CA3区神经元簇发放电持续时间及簇发放电内动作电位的个数与CA1及DG区相比有显著的统计学差异;剪断CA3与CA1间的Schaffer氏纤维后,CA1区的电活动消失,CA3区仍有同步放电,且其自发同步放电的频率与对照组相比无显着改变,但其簇发放电持续时间及簇发放电内动作电位的个数明显降低（P<0.05）。 结论 成功地在多电极上记录到急性海马切片自发、同步、癫痫样电活动;其中CA3区神经元兴奋性最高;在低镁灌流下自发癫痫样电活动起源于CA3区,在剪断Schaffer氏侧支后CA3区神经元群体同步放电的频率的频率没有显着变化。     

低镁诱发急性海马切片高兴奋性的多电极记录

目的 多电极记录技术在脑片研究的应用已经远远超过了10年,然而该技术却没有广泛地用于癫痫领域的研究。用经典的致痫剂低镁人工脑脊液灌流大鼠急性海马切片,诱导产生癫痫样电活动并用多电极记录技术对其放电特征及内部传导方式进行分析。 方法 用多电极阵列持续记录灌流低镁人工脑脊液后海马各区域的放电情况,并比较切断CA3与CA1区域间的Schaffer氏纤维后各区的放电情况。 结果 在急性海马切片上诱导出自发、同步、癫痫样电活动;CA3区神经元簇发放电持续时间及簇发放电内动作电位的个数与CA1及DG区相比有显著的统计学差异;剪断CA3与CA1间的Schaffer氏纤维后,CA1区的电活动消失,CA3区仍有同步放电,且其自发同步放电的频率与对照组相比无显着改变,但其簇发放电持续时间及簇发放电内动作电位的个数明显降低（P<0.05）。 结论 成功地在多电极上记录到急性海马切片自发、同步、癫痫样电活动;其中CA3区神经元兴奋性最高;在低镁灌流下自发癫痫样电活动起源于CA3区,在剪断Schaffer氏侧支后CA3区神经元群体同步放电的频率的频率没有显着变化。     

Magnesium deprivation decreases cellular reduced glutathione and causes oxidative neuronal death in murine cortical cultures

The vulnerability of cultured cortical neurons to oxidative injury is an inverse function of the extracellular Mg2+ concentration. In order to test the hypothesis that depolarization-enhanced release of reduced glutathione (GSH) contributes to this phenomenon, we assessed the effect of Mg2+ deprivation on cellular and medium glutathione levels. Incubation of mixed neuronal and glial cultures in Mg2+-free medium resulted in a decline in cellular total glutathione (GSx) within 8 h, without change in oxidized glutathione (GSSG); no effect was seen in pure glial cultures. This decrease in cellular GSx was associated with a progressive increase in GSx but not GSSG in the culture medium. Cellular GSH loss was not attenuated by concomitant treatment with antioxidants (ascorbate, Trolox, or deferoxamine), but was prevented by the NMDA receptor antagonist MK-801. Mg2+ deprivation for over 24 h produced neuronal but not glial death, with release of about 40% of neuronal lactate dehydrogenase by 48-60 h. Most of this cytotoxicity was prevented by treatment with either antioxidants or MK-801. These results suggest that Mg2+ deprivation causes release of neuronal reduced glutathione via a mechanism involving excessive NMDA receptor activation. If prolonged, cellular GSH depletion ensues, leading to oxidative neuronal death.     

Transient epileptiform signaling during neuronal network development: regulation by external stimulation and bimodal GABAergic activity

A predominance of excitatory activity, with protracted appearance of inhibitory activity, accompanies cortical neuronal development. It is unclear whether or not inhibitory neuronal activity is solicited exclusively by excitatory neurons or whether the transient excitatory activity displayed by developing GABAergic neurons contributes to an excitatory threshold that fosters their conversion to inhibitory activity. We addressed this possibility by culturing murine embryonic neurons on multi-electrode arrays. A wave of individual 0.2–0.4 mV signals (“spikes”) appeared between approx. 20–30 days in culture, then declined. A transient wave of high amplitude (>0.5 mV) epileptiform activity coincided with the developmental decline in spikes. Bursts (clusters of ≥3 low-amplitude spikes within 0.7 s prior to returning to baseline) persisted following this decline.     

Effects of retigabine on rhythmic synchronous activity of human neocortical slices

The antiepileptic effects of the novel antiepileptic drug retigabine (D-23129) [N-(2-amino-4-(4-flurobenzylamino)phenyl) carbamid acid ethyl ester] were tested in neocortical slice preparations (n=23) from 17 patients (age, 3-42 years) who underwent surgery for the treatment of intractable epilepsy. Epileptiform events consisted of spontaneously occurring rhythmic sharp waves, as well as of epileptiform field potentials (EFP) elicited by superfusion with Mg(2+)-free solution without or with addition of 10 micromol/l bicuculline. (1) Spontaneous rhythmic sharp waves (n=6), with retigabine application, the repetition rate was decreased down to 12-47% of initial value (10 micromol/l, n=3) after 180 min or suppressed completely within 12 min (50 micromol/l, n=3). (2) Low Mg(2+) EFP (n=9), with retigabine application, the repetition rate was decreased down to 50 and 65% of initial value (10 micromol/l; n=2) after 180 min or suppressed completely after 9-55 min (10, 50 and 100 micromol/l; n=2 in each case). In one slice only a transient reduction of the repetition rate was seen with 10 micromol/l retigabine. (3) Low Mg(2+) EFP with addition of bicuculline (n=8), with retigabine application, the repetition rate was decreased down to 12-55% of initial value (10 micromol/l; n=4) after 180 min or suppressed completely after 6-30 min (50 and 100 micromol/l; n=2 in each case). The depressive effect of retigabine was reversible in all but one slice. The results show a clear antiepileptic effect of retigabine in human neocortical slices on spontaneously occurring rhythmic sharp waves and different types of induced seizure activity.     

Region-specific modulation of limbic seizure susceptibility by ovarian steroids

Gonadal steroid hormones can markedly affect seizure susceptibility. Ovariohysterectomized female rats given ovarian steroid hormone supplements were used to evaluate the effects of ovarian steroids on epileptiform activity in hippocampal slices in vitro and on flurothyl-induced seizures in vivo. Seizure susceptibility was compared in the entorhinal cortex (EC) and CA1 regions of the hippocampus perfused with Mg(2+)-free medium, which leads to epileptiform discharges caused by a relief of voltage-dependent NMDA receptor block. After in vivo treatment with 500 microg of progesterone for 2 h prior to slice preparation, the latency to onset of low Mg(2+)-induced epileptiform activity of slices was significantly prolonged compared to slices from controls. In contrast, progesterone replacement accelerated the development of epileptiform activity in the CA1 region. Neither estrogen alone (2 x 2 microg of estradiol benzoate, 48 and 24 h prior to the experiment), nor a combined treatment with estrogen plus progesterone, significantly affected seizure susceptibility in either CA1 or the EC. There were no consistent effects of estrogen or progesterone, alone or in combination, on flurothyl-induced seizures in vivo. The data suggest that in vitro, progesterone alters seizure susceptibility in a site- and seizure model-specific fashion. The differential effects of progesterone may be due to differential expression of progesterone receptor isoforms or metabolites in specific brain areas suggesting that selective modulation of NMDA receptor-dependent epileptiform activity may play a role in hormonal effects on epileptogenesis.     

Iron-induced experimental cortical seizures: Electroencephalographic mapping of seizure spread in the subcortical brain areas

The iron-induced model of post-traumatic chronic focal epilepsy in rats was studied by depth-electrode mapping to investigate the spread of epileptiform activity into subcortical brain structures after its onset in the cortical epileptic focus. Electrical seizure activity was recorded in the hippocampal CA1 and CA3 areas, amygdala and caudate-putamen, in rats with iron-induced chronic cortical focal epilepsy. These experiments showed that the epileptiform activity with its onset in the cortical focus synchronously propagated into the studied subcortical brain areas. Seizure behaviours seemed to increase in correspondence with the spread of the epileptic electrographic activity in subcortical areas. Comparison of the cortical focus electroencephalographic and associated multiple-unit action potential recordings with those from the subcortical structures showed that the occurrence and evolution of the epileptiform activity in the subcortical structures were in parallel with that in the cortical focus. The intracerebral anatomic progression and delineation of seizure spread (mapped by field potential (EEG) and multiple-unit action potentials (MUA) recordings) indicated participation of these regions in the generalization of seizure activity in this model of epilepsy. The seizure-induced activation of the hippocampus appeared to evolve into an epileptic focus independent of the cortical focus. The present study demonstrates the propagation of epileptic activity from the cortical focus into the limbic and basal ganglia regions. Treatment of iron-induced epileptic rats with ethosuximide, an anti-absence drug, resulted in suppression of the epileptiform activity in the cortical focus as well as in the subcortical brain areas.