Epileptogenesis induces long-term alterations in intracellular calcium release and sequestration mechanisms in the hippocampal neuronal culture model of epilepsy

Calcium and calcium-dependent processes have been hypothesized to be involved in the induction of epilepsy. It has been shown that epileptic neurons have altered calcium homeostatic mechanisms following epileptogenesis in the hippocampal neuronal culture (HNC) and pilocarpine models of epilepsy. To investigate the mechanisms causing these alterations in [Ca2+]i homeostatic processes following epileptogenesis, we utilized the HNC model of in vitro 'epilepsy' which produces spontaneous recurrent epileptiform discharges (SREDs). Using [Ca2+]i imaging, studies were initiated to evaluate the mechanisms mediating these changes in [Ca2+]i homeostasis. 'Epileptic' neurons required much longer to restore a glutamate induced [Ca2+]i load to baseline levels than control neurons. Inhibition of Ca2+ entry through voltage and receptor gated Ca2+ channels and stretch activated Ca2+ channels had no effect on the prolonged glutamate induced increase in [Ca2+]i in epileptic neurons. Employing thapsigargin, an inhibitor of the sarco/endoplasmic reticulum calcium ATPase (SERCA), it was shown that thapsigargin inhibited sequestration of [Ca2+]i by SERCA was significantly decreased in 'epileptic' neurons. Using Ca2+ induced Ca2+ release (CICR) cell permeable inhibitors for the ryanodine receptor (dantrolene) and the IP3 receptor (2-amino-ethoxydiphenylborate, 2APB) mediated CICR, we demonstrated that CICR was significantly augmented in the 'epileptic' neurons, and determined that the IP3 receptor mediated CICR was the major release mechanism altered in epileptogenesis. These data indicate that both inhibition of SERCA and augmentation of CICR activity contribute to the alterations accounting for the impaired calcium homeostatic processes observed in 'epileptic' neurons. The results suggest that persistent changes in [Ca2+]i levels following epileptogenesis may contribute to the long-term plasticity changes manifested in epilepsy and that understanding the basic mechanisms mediating these changes may provide an insight into the development of novel therapeutic approaches to treat epilepsy and prevent or reverse epileptogenesis.     

Induction of spontaneous recurrent epileptiform discharges causes long-term changes in intracellular calcium homeostatic mechanisms

Calcium and calcium-dependent systems have been long implicated in the induction of epilepsy. We have previously observed that intracellular calcium ([Ca2+]i) levels remain elevated in cells undergoing epileptogenesis in the hippocampal neuronal culture (HNC) model. In this study, we employed the hippocampal neuronal culture (HNC) model of in vitro 'epilepsy' which produces spontaneous recurrent epileptiform discharges (SREDs) for the life of the neurons in culture to investigate alterations in [Ca2+]i homeostatic mechanisms that may be associated with the 'epileptic' phenotype. [Ca2+]i imaging fluorescence microscopy was performed on control and 'epileptic' neurons with two different fluorescent dyes ranging from high to low affinities for [Ca2+]i. We measured baseline [Ca2+]i levels and the ability to restore resting [Ca2+]i levels after a brief 2-min exposure to the excitatory amino acid glutamate in control neurons and neurons with SREDs. Neurons manifesting SREDs had statistically significantly higher baseline [Ca2+]i levels that persisted for the life of the culture. In addition, the 'epileptic' phenotype was associated with an inability to rapidly restore [Ca2+]i levels to baseline following a glutamate induced [Ca2+]i load. The use of the low affinity dye Fura-FF demonstrated that the difference in restoring baseline [Ca2+]i levels was not due to saturation of the high affinity dye Indo-1, which was utilized for evaluating the [Ca2+]i kinetics at lower [Ca2+]i levels. Peak [Ca2+]i levels in response to glutamate were the same in both 'epileptic' and control neurons. While [Ca2+]i levels recovered in approximately 30 min in control cells, it took more than 90 min to reach baseline levels in cells manifesting SREDs. Alterations of [Ca2+]i homeostatic mechanisms observed with the 'epileptic' phenotype were shown to be independent of the presence of continuous SREDs and persisted for the life of the neurons in culture. Epileptogenesis was shown not to affect the degree or duration of glutamate induced neuronal depolarization in comparing control and 'epileptic' neurons. The results indicate that epileptogenesis in this in vitro model produced long-lasting alterations in [Ca2+]i regulation that may underlie the 'epileptic' phenotype and contribute to the persistent neuroplasticity changes associated with epilepsy.     

Epileptogenesis after Experimental Focal Cerebral Ischemia

Cerebrovascular diseases are one of the most common causes of epilepsy in adults, and the incidence of stroke-induced epileptogenesis is increasing as the population ages. The mechanisms that lead to stroke-induced epileptogenesis in a subpopulation of patients, however, are still poorly understood. Recent advances in inducing epileptogenesis in rodent focal ischemia models have provided tools that can be used to identify the risk factors and neurobiologic changes leading to development of epilepsy after stroke. Here we summarize data from models in which epileptogenesis has been studied after focal ischemia; photothrombosis, middle cerebral artery (MCA) occlusion with filament, and endothelin-1-induced MCA occlusion. Analysis of the data indicates that neurobiologic changes occurring during stroke-induced epileptogenesis share some similarities to those induced by status epilepticus or traumatic brain injury. Special issue dedicated to Dr. Simo S. Oja     

Mitochondrial matters of the brain: mitochondrial dysfunction and oxidative status in epilepsy

Epilepsy is a neurological disorder characterized by spontaneous, recurrent and paroxysmal cerebral discharge, clinically leading to persistent alterations in function and morphology of neurons. Oxidative stress is one of possible mechanisms in the pathogenesis of epilepsy. Oxidative stress resulting from mitochondrial dysfunction gradually disrupts the intracellular calcium homeostasis, which modulates neuronal excitability and synaptic transmission making neurons more vulnerable to additional stress, and leads to neuronal loss in epilepsy. In addition, the high oxidative status is associated with the severity and recurrence of epileptic seizure. Hence, treatment with antioxidants is critically important in epileptic patients through scavenging the excessive free radicals to protect the neuronal loss. In this review, we reviewed the recent findings that focus on the role for antioxidants in prevention of mitochondrial dysfunction and the correlation between oxidative status and disease prognosis in patients with epilepsy.     

膜超极化激活离子通道及其靶向抗癫痫药物研究进展

癫痫是一种较为常见的神经系统疾病,主要以大量神经元同步异常放电为特征。目前普遍认为,神经元或神经网络兴奋性和抑制性 电信号传输的失衡,是癫痫发病的最根本原因。现有的抗癫痫药物主要以钠离子通道、钙离子通道、钾离子通道、谷氨酸受体和γ-氨基丁 酸离子通道为靶点,但接受这些药物治疗后,仍有近1/3的病人无法控制癫痫发作。因此,抗癫痫药物的研发亟需新靶点和新思路。许多 研究证据表明,膜超极化激活离子通道的基因突变可以导致遗传型癫痫的发作,且在脑部损伤后,膜超极化激活离子通道会发生表达水平、 通道生物物理学性质及通道亚基构成的改变,从而增加神经元和神经网络兴奋性,促使癫痫发病。故近年来,膜超极化激活离子通道及其 靶向抗癫痫药物研究引起人们广泛关注。综述膜超极化激活离子通道与癫痫发病之间的关系,并探讨以膜超极化激活离子通道为靶点进行 抗癫痫药物开发和治疗的可行性。     

Glutamate uptake in blood platelets from epileptic patients

Glutamate, the major excitatory amino acid neurotransmitter, is involved in epileptogenesis and initiation and spread of seizures. We studied glutamate uptake into blood platelets from patients with distinct epileptic syndromes: included were 20 patients with temporal lobe epilepsy and hippocampal sclerosis (TLE+HS), 20 with juvenile myoclonus epilepsy (JME) and 20 healthy volunteers matched for age and sex. The affinity of glutamate for the transporters was highest in patients with TLE+HS, but the maximal velocity of transport was highest in controls. There were no differences in the plasma levels of glutamate. Carbamazepine (CBZ), valproate (VPA) and lamotrigine (LTG) did not affect the uptake in vitro. The alterations observed in the uptake of glutamate in TLE+HS patients may reflect an up-regulated uptake of glutamate in the brain.     

Epileptiform postsynaptic currents in primary culture of rat cortical neurons: Calcium mechanisms

In this study we demonstrate that the primary culture of rat cortical neurons is a convenient model for investigations of epileptogenesis mechanisms and specifically, of the postsynaptic epileptiform currents (EC) reflecting periodical asynchronous glutamate release. In particular, we have revealed that in primary culture of cortical neurons EC can appear spontaneously or can be triggered by the withdrawal of magnesium block of NMDA receptor channels or by shutting down GABAergic inhibition. EC were found to depend on intracellular calcium oscillations. The secondary calcium release from intracellular stores was needed for EC synchronization. EC were suppressed by the influences causing either neuronal calcium overload or decrease of intracellular calcium concentration. Calcium entry into neurons in the case of NMDA receptor hyperactivation or in the case of calcium ionophore ionomycin treatment eliminated EC. The suppression of EC also occurred after a decrease of intracellular calcium concentration induced by BAPTA loaded into the neurons or by stimulation of calcium removal from cells via Na⁺/Ca²⁺ exchanger by 1 nM ouabain. Partial dependence of EC on action potential generation was found. Thus, EC in neurons are activated by intracellular periodic calcium waves within a limited concentration window.     

Alterations in calcium-mediated signal transduction after traumatic injury of cortical neurons

Calcium influx and elevation of intracellular free calcium ([Ca2+]i), with subsequent activation of degradative enzymes, is hypothesized to cause cell injury and death after traumatic brain injury. We examined the effects of mild-to-severe stretch-induced traumatic injury on [Ca2+]i dynamics in cortical neurons cultured on silastic membranes. [Ca2+]i was rapidly elevated after injury, however, the increase was transient with neuronal [Ca2+]i returning to basal levels by 3 h after injury, except in the most severely injured cells. Despite a return of [Ca2+]i to basal levels, there were persistent alterations in calcium-mediated signal transduction through 24 h after injury. [Ca2+]i elevation in response to glutamate or NMDA was enhanced after injury. We also found novel alterations in intracellular calcium store-mediated signaling. Neuronal calcium stores failed to respond to a stimulus 15 min after injury and exhibited potentiated responses to stimuli at 3 and 24 h post-injury. Thus, changes in calcium-mediated cellular signaling may contribute to the pathology that is observed after traumatic brain injury.     

The potential role of mitochondrial dysfunction in seizure-associated cell death in the hippocampus and epileptogenesis

Epilepsy is considered one of the most common neurological disorders worldwide. The burst firing neurons associated with prolonged epileptic discharges could lead to a large number of changes with events of cascades at the cellular level. From its role as the cellular powerhouse, mitochondria also play a crucial role in the mechanisms of cell death. Emerging evidence has shown that prolonged seizures may result in mitochondrial dysfunction and increase of oxidative and nitrosative stress in the hippocampus that precede neuronal cell death and cause subsequent epileptogenesis. The selective dysfunction of mitochondrial respiratory chain Complex I has been suggested to be a biochemical hallmark of seizure-induced neuronal cell death and epileptogenesis. Therefore, protection of mitochondria from bioenergetic failure and oxidative stress in the hippocampus may open a new vista to the development of effective neuroprotective strategies against seizure-induced brain damage and to the design of novel treatment perspectives against therapy-resistant forms of epilepsy.     

Phase-Dependent Astroglial Alterations in Li–Pilocarpine-Induced <Emphasis Type="Italic">Status Epilepticus</Emphasis> in Young Rats

Epilepsy prevalence is high in infancy and in the elderly population. Lithium–pilocarpine is widely used to induce experimental animal models of epilepsy, leading to similar neurochemical and morphological alterations to those observed in temporal lobe epilepsy. As astrocytes have been implicated in epileptic disorders, we hypothesized that specific astroglial changes accompany and contribute to epileptogenesis. Herein, we evaluated time-dependent astroglial alterations in the hippocampus of young (27-day-old) rats at 1, 14 and 56 days after Li–pilocarpine-induced status epilepticus (SE), corresponding to different phases in this model of epilepsy. We determined specific markers of astroglial activation: GFAP, S100B, glutamine synthetase (GS), glutathione (GSH) content, aquaporin-4 (AQP-4) and potassium channel Kir 4.1; as well as epileptic behavioral, inflammatory and neurodegenerative changes. Phase-dependent signs of hippocampal astrogliosis were observed, as demonstrated by increments in GFAP, S100B and GS. Astrocyte dysfunction in the hippocampus was characterized, based on the decrease in GSH content, AQP-4 and Kir 4.1 channels. Degenerating neurons were identified by Fluoro-Jade C staining. We found a clear, early (at SE1) and persistent (at SE56) increase in cerebrospinal fluid (CSF) S100B levels. Additionally, serum S100B was found to decrease soon after SE induction, implicating a rapid-onset increase in the CSF/serum S100B ratio. However, serum S100B increased at SE14, possibly reflecting astroglial activation and/or long-term increase in cerebrovascular permeability. Moreover, we suggest that peripheral S100B levels may represent a useful marker for SE in young rats and for follow up during the chronic phases of this model of epilepsy. Together, results reinforce and extend the idea of astroglial involvement in epileptic disorders.     

Calcium: a role for neuroprotection and sustained adaptation

Sustained increases in intracellular calcium following prolonged seizures or other neurological insults have been thought to be responsible for neuronal cell death for well over two decades. For example, a seizure or a stroke can lead to excessive release of glutamate, an endogenous excitotoxin. Overactivation of receptors that interact with glutamate will raise calcium levels to stimulate a variety of signaling pathways that can impair neuronal respiration and eventually kill neurons. On the contrary, recent evidence shows that under numerous conditions calcium can prevent neurons from dying. Experimental epilepsy and ischemia models show that protection of neurons appears to depend upon the age of the animal, the amount and route of calcium elevation, timing of initial insults, and brain regions involved. This review will discuss novel findings on the protective signaling role of calcium under a wide range of pathological conditions.     

Calcium oscillations in neocortical astrocytes under epileptiform conditions.

Morphological and functional alterations in astrocytic glia are often found in epileptic syndromes, although the exact role of astrocytes in epilepsy is poorly understood. During calcium imaging of epileptiform events in juvenile neocortical slices we previously discovered cells with spontaneous oscillations in their intracellular free calcium concentration ([Ca(2+)](i)). We have now characterized these oscillations using two in vitro models of epilepsy and find that they are produced by astrocytes. Astrocytic oscillations are widespread throughout the imaged territories, are remarkably regular and have long periods, averaging 100 s, which become shorter during development. Astrocytic oscillations are uncorrelated among themselves and with epileptiform events, are blocked by internal release antagonists and are stimulated by caffeine. Astrocytic calcium oscillations could mediate reactive astrogliosis, contribute to the pathogenesis of chronic epileptic syndromes, and be used as a diagnostic test for epileptic tissue.     

Mitochondrial dysfunction and oxidative stress: a contributing link to acquired epilepsy?

Mitochondrial dysfunction and oxidative stress contribute to several neurologic disorders and have recently been implicated in acquired epilepsies such as temporal lobe epilepsy (TLE). Acquired epilepsy is typically initiated by a brain injury followed by a "latent period" whereby molecular, biochemical and other cellular alterations occur in the brain leading to chronic epilepsy. Mitochondrial dysfunction and oxidative stress are emerging as factors that not only occur acutely as a result of precipitating injuries such as status epilepticus (SE), but may also contribute to epileptogenesis and chronic epilepsy. Mitochondria are the primary site of reactive oxygen species (ROS) making them uniquely vulnerable to oxidative damage that may affect neuronal excitability and seizure susceptibility. This mini-review provides an overview of evidence suggesting the role of mitochondrial dysfunction and oxidative stress as acute consequences of injuries that are known to incite chronic epilepsy and their involvement in the chronic stages of acquired epilepsy.     

Sonic hedgehog is a regulator of extracellular glutamate levels and epilepsy

Sonic hedgehog (Shh), both as a mitogen and as a morphogen, plays an important role in cell proliferation and differentiation during early development. Here, we show that Shh inhibits glutamate transporter activities in neurons, rapidly enhances extracellular glutamate levels, and affects the development of epilepsy. Shh is quickly released in response to epileptic, but not physiological, stimuli. Inhibition of neuronal glutamate transporters by Shh depends on heterotrimeric G protein subunit Gα_i and enhances extracellular glutamate levels. Inhibiting Shh signaling greatly reduces epileptiform activities in both cell cultures and hippocampal slices. Moreover, pharmacological or genetic inhibition of Shh signaling markedly suppresses epileptic phenotypes in kindling or pilocarpine models. Our results suggest that Shh contributes to the development of epilepsy and suppression of its signaling prevents the development of the disease. Thus, Shh can act as a modulator of neuronal activity, rapidly regulating glutamate levels and promoting epilepsy.     

Calcium oscillations in neocortical astrocytes under epileptiform conditions

Morphological and functional alterations in astrocytic glia are often found in epileptic syndromes, although the exact role of astrocytes in epilepsy is poorly understood. During calcium imaging of epileptiform events in juvenile neocortical slices we previously discovered cells with spontaneous oscillations in their intracellular free calcium concentration ([Ca²⁺]_i). We have now characterized these oscillations using two in vitro models of epilepsy and find that they are produced by astrocytes. Astrocytic oscillations are widespread throughout the imaged territories, are remarkably regular and have long periods, averaging 100 s, which become shorter during development. Astrocytic oscillations are uncorrelated among themselves and with epileptiform events, are blocked by internal release antagonists and are stimulated by caffeine. Astrocytic calcium oscillations could mediate reactive astrogliosis, contribute to the pathogenesis of chronic epileptic syndromes, and be used as a diagnostic test for epileptic tissue. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 45–55, 2002     

Homeostatic regulation mechanism of spontaneous firing determines glutamate responsiveness in the midbrain dopamine neurons

Autonomous tonic firing of the midbrain dopamine neuron is essential for maintenance of ambient dopamine level in the brain, in which intracellular Ca²⁺ concentration ([Ca²⁺]c) plays a complex but pivotal role. However, little is known about Ca²⁺ signals by which dopamine neurons maintain an optimum spontaneous firing rate. In the midbrain dopamine neurons, we here show that spontaneous firing evoked [Ca²⁺]c changes in a phasic manner in the dendritic region but a tonic manner in the soma. Tonic levels of somatic [Ca²⁺]c strictly tallied with spontaneous firing rates. However, manipulatory raising or lowering of [Ca²⁺]c with caged compounds from the resting firing state proportionally suppressed or raised spontaneous firing rate, respectively, suggesting presence of the homeostatic regulation mechanism for spontaneous firing rate via tonic [Ca²⁺]c changes of the soma. More importantly, abolition of this homeostatic regulation mechanism significantly exaggerated the responses of tonic firings and high-frequency phasic discharges to glutamate. Therefore, we conclude that this Ca²⁺-dependent homeostatic regulation mechanism is responsible for not only maintaining optimum rate of spontaneous firing, but also proper responses to glutamate. Perturbation of this mechanism could cause dopamine neurons to be more vulnerable to glutamate and Ca²⁺ toxicities.     

Variations of ATP and its metabolites in the hippocampus of rats subjected to pilocarpine-induced temporal lobe epilepsy

Although purinergic receptor activity has lately been associated with epilepsy, little is known about the exact role of purines in epileptogenesis. We have used a rat model of temporal lobe epilepsy induced by pilocarpine to study the dynamics of purine metabolism in the hippocampus during different times of status epilepticus (SE) and the chronic phase. Concentrations of adenosine 5′-triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine in normal and epileptic rat hippocampus were determined by microdialysis in combination with high-performance liquid chromatography (HPLC). Extracellular ATP concentrations did not vary along 4 h of SE onset. However, AMP concentration was elevated during the second hour, whereas ADP and adenosine concentrations augmented during the third and fourth hour following SE. During chronic phase, extracellular ATP, ADP, AMP, and adenosine concentrations decreased, although these levels again increased significantly during spontaneous seizures. These results suggest that the increased turnover of ATP during the acute period is a compensatory mechanism able to reduce the excitatory role of ATP. Increased adenosine levels following 4 h of SE may contribute to block seizures. On the other hand, the reduction of purine levels in the hippocampus of chronic epileptic rats may result from metabolic changes and be part of the mechanisms involved in the onset of spontaneous seizures. This work provides further insights into purinergic signaling during establishment and chronic phase of epilepsy.