Cell Tissue Res (2014) 357:385–393 DOI 10.1007/s00441-014-1849-1

REVIEW

Calcium signaling and epilepsy Ortrud K. Steinlein

Received: 28 August 2013 / Accepted: 13 February 2014 / Published online: 11 April 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Calcium signaling is involved in a multitude of physiological and pathophysiological mechanisms. Over the last decade, it has been increasingly recognized as an important factor in epileptogenesis, and it is becoming obvious that the excess synchronization of neurons that is characteristic for seizures can be linked to various calcium signaling pathways. These include immediate effects on membrane excitability by calcium influx through ion channels as well as delayed mechanisms that act through G-protein coupled pathways. Calcium signaling is able to cause hyperexcitability either by direct modulation of neuronal activity or indirectly through calciumdependent gliotransmission. Furthermore, feedback mechanisms between mitochondrial calcium signaling and reactive oxygen species are able to cause neuronal cell death and seizures. Unravelling the complexity of calcium signaling in epileptogenesis is a daunting task, but it includes the promise to uncover formerly unknown targets for the development of new antiepileptic drugs. Keywords Calcium signaling . Epilepsy . Gliotransmission . Seizures . Hyperexcitability

Introduction Most physiological functions depend on a multitude of signaling pathways that govern cellular activities, coordinate cell actions, and exchange information between cells. None of these signaling pathways would be able to function properly without the small but multi-functional calcium ions. Calcium O. K. Steinlein (*) Institute of Human Genetics, University Hospital Munich, Ludwig-Maximilians University Munich, Goethestr 29, 80336 Munich, Germany e-mail: [email protected]

ions can act as second messengers and, by doing this, they are involved in many different signaling pathways. They are able to directly bind to ion channels and modulate their activity or to act indirectly, for example by binding to calmodulin and thereby changing the activity of G protein-activated enzymes. Many of these G-protein-coupled pathways are either directed at key transmembrane receptors and ion channels which are functionally altered by phosphorylation or dephosphorylation, or regulate transcription and translation of different proteins. Cellular recruitment of calcium ions is possible from many sources, such as by an influx through voltage-dependent channels or by release from the smooth endoplasmic reticulum through calcium permeable channels (Bading 2013; Greer and Greenberg 2008; Hartmann and Konnerth 2005; Hawk and Abel 2011; Tanaka 2001). Calcium-dependent signaling pathways interact with neuronal activity in many ways and it is therefore not surprising that calcium ions are regarded as an important factor in both epileptogenesis and seizure generation (Fig. 1). The term “epilepsy” describes a large group of heterogeneous disorders that all have in common episodic attacks of focal or generalized neuronal hyperexcitability. With an incidence of 0.5–1 % epilepsy is one of the most common neurological disorders, but treatment is only successful in a limited number of patients. It is therefore important to find new approaches by which seizure frequency in patients can be controlled. In recent years. calcium signaling has become a promising new target for the development of antiepileptic drugs. Some of the recently introduced drugs such as levetiracetam already exhibit at least part of their action through the modulation of intracellular calcium influx (Niespodziany et al. 2001; Ozcan and Ayar 2012; Takahashi et al. 2010). The examples described in the following sections demonstrate that the many different mechanisms by which calcium signaling is involved in neuronal excitability and seizure generation present numerous additional targets for anti-epileptic drug development (Table 1).

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Fig. 1 Epileptogenesis and seizure generation. The term epileptogenesis describes the process by which epilepsy develops in the brain. Once established, episodic seizure generation occurs sporadically

Epilepsy

Seizure generation

Epileptogenesis

Initiation

Calcium signaling and hyperexcitable neurons The impact calcium signaling has on neuronal excitability can be either instant or delayed. Instant effects are, for example, caused by the activation of calcium-dependent potassium channels or by inactivation of voltage-dependent calcium channels (Berkefeld et al. 2006; Hirschberg et al. 1999). This type of signaling tends to remain restricted to a smaller group of neurons or a localized brain area because the availability of calcium ions is tightly regulated. Calcium signaling that effects the phosphorylation or dephosphorylation of ion channels has longer lasting effects, and is especially important for the modulation of neuronal excitability and synaptic plasticity. The spatial spreading of this type of calcium signaling depends on the mobility of the second messengers and enzymes that are activated by the signaling. It can spread widely Table 1 Nomenclature of voltage-gated calcium channels Type

Subunit

Gene (human)

L-type

Cav1.1

CACNA1S

Cav1.2 Cav1.3 Cav1.4 Cav2.1 Cav2.2 Cav2.3 Cav3.1 Cav3.2 Cav3.3

CACNA1C CACNA1D CACNA1F CACNA1A CACNA1B CACNA1E CACNA1G CACNA1H CACNA1I

P-type/Q-type N-type R-type T-type

a

Gene (mouse)a

Cacna1a

Cacna1g

Symbols for murine channels are only listed if mentioned in the main text. Source: http://www.iuphar-db.org, http://www.nlm.nih.gov/cgi/ mesh

First seizure

Recurrent seizures

when these molecules are diffusible or remain strictly localized if calcium acts on membrane-bound molecules. Even longer lasting effects that might continue through life are possible when calcium signaling affects gene expression by the modulation of transcriptional or translational processes (Abel et al. 1997; Costa-Mattioli et al. 2009; Daoudal and Debanne 2003; Hulme et al. 2003; Lisman et al. 2002; Shalin et al. 2006; Tostevin et al. 2007). Epilepsy caused by brain trauma represents perhaps the most thoroughly studied relationship between calcium signaling and epileptogenesis. It has been shown that trauma produces an elevation of intracellular calcium that in its duration is directly correlated to the extent of the injury (Gurkoff et al. 2012; Shahlaie et al. 2010; Weber et al. 1999). The marked calcium influx is part of the glutamate-triggered neuronal injury process observed after brain trauma or in neurodegenerative diseases (Arundine and Tymianski 2003). The overactivation of NMDA receptors by glutamate causes an excessive influx of calcium which overwhelms calciumregulatory mechanisms. The resulting cell damage might render neuronal networks vulnerable to seizures and even resistant to antiepileptic drugs. The elevated intracellular calcium levels are known to also substantially increase the transamidating activity of tTG (tissue transglutaminase) (Smethurst and Griffin 1996). Overexpression of tTG in a transgenic mouse model showed that tTG is likely to play a role in glutamate-induced neurotoxicity. The mechanism by which tTG exerts this effect is not known, but both an enhancement of seizure activity and a facilitation of downstream apoptotic processes are discussed as possible explanations (Tucholski et al. 2006). It is evident that calcium ions are crucial for this effect, because, in normal brain with its low calcium levels, tTG shows only a scarce activity, even if overexpressed as was done in the above-

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mentioned transgenic mouse model. However, the transamidating activity of tTG is no longer inhibited by GTP binding once calcium levels are elevated, for example by some type of head trauma. Thus, tTG only facilitates cell death in situations that cause an intracellular calcium influx as is typical for seizures and other neurological disorders (Achyuthan and Greenberg 1987; Smethurst and Griffin 1996; Tucholski et al. 2006). Ictal neuronal hyperactivity can either remain localized (focal seizure) or rapidly spread through brain (generalized seizure). Studies with animal models have shown that various calcium channels are crucial for seizure propagation. These channels mediate the entry of calcium ions into neurons in response to membrane depolarization. This in turn modulates a number of essential neuronal responses including the control of neuronal excitability. In GAERS rats, which are an established model for studying the mechanisms and genetics of absence epilepsy, the development of seizures is accompanied by an increase in T-type calcium currents. This increase is putatively due to a rising expression of Cav3.1 (CACNA1G) and Cav3.2 (CACNA1H) T-type calcium channels in reticular neurons. This rise in expression precedes the onset of seizures, an observation that has led to the speculation that the calcium channels might facilitate epileptogenesis by an alteration of neuronal network dynamics (Khosravani and Zamponi 2006; Thomas and Grisar 2000). However, an increase in calcium channel expression can not only be observed during the initial phase of epileptogenesis but also after the onset of seizures. It has been shown that a single episode of status epilepticus is sufficient to cause a lasting increase in T-type calcium channel activity. Thus, once established, the neuronal epileptic activity seems to be able to propagate itself by keeping calcium channel expression enhanced (Su et al. 2002).

Role of astrocyte calcium signaling in seizure propagation Astrocytic glial cells (astrocytes) are the most abundant cell type in brain. Their importance for brain function has long been underestimated, and they have been regarded mostly as gap-fillers that might have a role in structural maintenance and tissue damage repair. However, this view has changed dramatically since the mid-1990s. It is now known that astrocytes perform many functions and are especially involved in the maintenance of extracellular ion balance. They are also known to be active players in synaptic transmission. It should therefore not be surprising that astrocytes are also involved in epileptogenesis. However, their emerging importance for seizure generation, although not yet fully understood, vastly exceeds all expectations. It is highly likely that they do not only participate in this process but are also involved in it from the very beginning (Blackburn et al. 2009; Maragakis and Rothstein 2006; Seifert et al. 2009).

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Seizures arise when groups of neurons show excess synchronization that produces hyperexcitability. Once this happens, an epileptogenic focus is born, with the potential that the hyperexcitability might spread to larger portions of the brain (secondary generalization). The key question remains: what causes the initial imbalance between inhibitory and excitatory activities that functions as the starting point of the ictal event. Recent studies have collected an increasing body of evidence that strongly implicates that astrocytes are key players in these mechanisms. At first, research focused on the role astrocytes have in the regulation of extracellular potassium ions. Increased extracellular potassium levels are typically found in epileptic tissue during a seizure, and astrocytes are able to remove these ions mainly by their Kir4.1 channels (David et al. 2009; Dietzel et al. 1989). These inward rectifying potassium channels have a channel conductance that is proportional to the extracellular potassium concentration (Ransom and Sontheimer 1995). They also show a high open probability at resting potentials and astrocytes are therefore well suited to buffer large amounts of potassium ions (Higashi et al. 2001; Li et al. 2001; Newman 1993; Poopalasundaram et al. 2000). It is therefore easy to imagine that an impaired astrocytic potassium removal mechanism could facilitate or even initiate seizures (Pedley et al. 1976; Wallraff et al. 2006). However, convincing evidence is still missing that such a scenario indeed describes a major mechanism in epileptogenesis. The focus of research therefore shifted from potassium to calcium ions, and it is becoming obvious that the latter are a crucial component of many astrocyte-based mechanisms involved in seizure generation. Nearly 20 years ago, it had been shown that calcium ions are required for the release of glutamate or other neurotransmitters such as ATP or adenosine from astrocytes (Parpura et al. 1994). Subsequently, the neurotransmitter release causes increased activity of neighboring neurons (Fellin et al. 2004). This observation was one of the first indications that astrocytes have a more active role in neurotransmission than previously granted. It suggested that astrocytes might be able to support epileptogenesis, maybe even generate seizures by themselves. Furthermore, it pointed out the important role calcium ions have in these mechanisms. Astrocytes at the location of the ictal focus were shown to have elevated calcium levels just prior to the onset of a seizure (Aronica et al. 2011; Fellin et al. 2006; Tian et al. 2005; Ulas et al. 2000). The hypothesis that this astrocytic calcium elevation has an important role in seizure generation, rather than being a side effect of neuronal hyperactivity, is supported by the fact that ictal discharges failed to occur when calcium elevations in astrocytes were suppressed by intracellular application of the chelator BAPTA (1,2-bis(o-amino-phenoxy)ethane-N,N,N’,N’,tetraacetic acid). It was demonstrated that, in such an experimental setting, the forced subactivity of astrocytes was accompanied by a decrease in the number of nearby neurons that

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showed hyperactivity (Gomez-Gonzalo et al. 2010; Losi et al. 2010). Furthermore, astrocytes not only have a crucial role in initiating neuronal hyperactivity but are also seen to actively recruit additional neurons once the ictal process has been initiated. The progress of this process seems to depend on a kind of feedback mechanism in which elevated intracellular calcium levels cause the astrocytes to release glutamate or Dserine, transmitters which in turn cause additional neurons to engage in hyperactivity (Gomez-Gonzalo et al. 2010; Lee et al. 2007; Shigetomi et al. 2008). The seizure is likely to occur once a critical number of neurons has reached a hyperactive state (Gomez-Gonzalo et al. 2010). However, these investigations are not able to tell us what happens at the very start of seizure generation, i.e. elevated intracellular calcium levels in astrocytes or hyperactivity of a few neurons. A tentative answer in favor of the neurons-first hypothesis might be provided by the observation that seizures could be provoked in a tissue slice model after BAPTA blocking if a large NMDA (N-Methyl-D-aspartate ) stimulus was applied (Gomez-Gonzalo et al. 2010). The stimulus is likely to engage a high number of neurons in hyperactivity at once, inducing a seizure without the need for astrocyte feedback. This suggests that astrocytes are not an indispensable part of seizure generation. However, compared to the NMDA stimulus used in the tissue model, spontaneous seizure generation in a patient’s brain is likely to start more subtly and might therefore depend on astrocyte feedback to gain the necessary momentum to become a clinically recordable seizure (Kovacs et al. 2000). Calcium signaling in astrocytes is not only likely to be involved in seizure generation but seems also to be able to promote the opposite effect. A decrease of calcium in a defined extracellular space has been shown to initiate in astrocytes a calcium-caused ATP release through connexion 43 hemichannels. The released ATP activates neuronal P2Y1 receptors which then promote an inhibitory signal (Torres et al. 2012). This effect is interesting because extracellular calcium levels are known to decrease during an epileptic discharge. The subsequent ATP release from astrocytes might act as a control mechanism that prevents the further spreading of the epileptic activity (Torres et al. 2012). Obviously, the mechanisms induced by calcium signaling in astrocytes are highly complex and many more studies need to be conducted before we are able to fully understand their different roles in seizure generation (Carmignoto and Haydon 2012; Reato et al. 2012).

Acquired epilepsy, mitochondria and calcium signaling Mitochondria are not only the cells’ main source for adenosine triphosphate (ATP) but they also serve as important calcium buffers. The calcium ions are able to increase ATP production,

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and ATP is subsequently able to produce reactive oxygen species (Brookes et al. 2004). Under physiological conditions, both calcium ions and reactive oxygen species act as signaling molecules in mitochondrial pathways. Calcium overload, however, causes mitochondrial dysfunction, oxidative stress, and cell damage. This pathomechanism seems to be especially important for the development of acquired epilepsies (DeLorenzo et al. 2005). These are a heterogeneous group of seizure disorders that are caused by external or internal events such as brain trauma, stroke, infection, or tumors. In the initial phase, the brain insult is believed to cause a cascade of structural and molecular changes, including increased production of reactive oxygen species (Pitkanen and Lukasiuk 2009). The latter dramatically increase the cytosolic calcium levels, which by negative feedback are able to further propagate the production of reactive oxygen species (Perez-Campo et al. 1998). The elevated calcium levels overstimulate calcium signaling pathways in mitochondria, causing mitochondrial dysfunction and energy depletion (Kovacs et al. 2002; Kunz et al. 1999) (Fig. 1). The final outcome is believed to depend on the level calcium reaches during this initial phase of epileptogenesis. Moderate calcium levels might inflict long-lasting functional changes, for example, by initiating signaling processes that cause a modulation of neuronal plasticity through changes in gene expression or protein function (DeLorenzo et al. 2005; Morris et al. 1999). High calcium concentrations are even able to induce instant neuronal necrosis or delayed apoptosis. It is likely that calcium-mediated necrosis/apoptosis constitutes an important factor in early epileptogenesis (i.e. a cause of epilepsy). Furthermore, it might also serve as a mechanism that is initiated by seizures and serves as an exacerbating factor once epilepsy has entered the phase where spontaneous recurrent seizures occur (Patel 2004; Waldbaum and Patel 2010) (Fig. 2).

Mutations directly affecting calcium signaling cause absence epilepsy In the previous paragraphs, examples were discussed in which calcium signaling is mostly indirectly involved in epileptogenesis and seizure generation. However, there are a growing number of examples that demonstrate that aberrant calcium signaling can also be a primary cause of genetic epilepsies and other neurological disorders. Recent studies found mutations in several ion-conducting and auxiliary calcium channel subunits causing different types of disorders that in humans include mostly neurological phenotypes such as episodic ataxia type 2, spinocerebellar ataxia type 6, familial hemiplegic migraine, and different forms of epilepsy (Chen et al. 2003; Heron et al. 2004; Jodice et al. 1997; Ophoff et al. 1996). Mutations causing childhood absence epilepsy have so

Cell Tissue Res (2014) 357:385–393 Fig. 2 Schematic representation of the putative ROS–calcium feedback mechanism in acquired epilepsy. The increased levels of ROS that are produced after brain damage enhance cytosolic calcium levels. The calcium ions further increase ROS production by feedback mechanism, and overactive calcium signaling damages mitochondrial function. This in turn can cause cell death and, consequently, seizures. The latter are able to induce even more cellular damage by further increasing ROS and calcium levels. ROS reactive oxygen species

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Neurological insult

far been described for the CACNA1A, CACNA1G, and CACN A1H calcium channel genes (Chen et al. 2003; Heron et al. 2004; Imbrici et al. 2004; Jouvenceau et al. 2001; Liang et al. 2006; Singh et al. 2007; Vitko et al. 2007). Most of these mutations are private, i.e. they have been found in only a sporadic patient or a single family. Calcium channel mutations are a very rare cause of epilepsy, but it nevertheless is a most interesting one for studying basic mechanisms of epileptogenesis in model systems. These functional studies are often performed as heterologous expression experiments in different types of cell lines, and they offer a first insight into the changes the underlying mutations cause in calcium channel function. For example, most of the calcium channel mutations clustered in the intracellular I–II loop of CACNA1H were found to increase the ratio of membrane expression over total expression, an observation that suggests that these mutations affect receptor surface expression. With 20–60 %, the estimated increase in surface expression was substantial, and a calcium channel “gain-of-function” effect was suggested to be at least one of the main factors that contributed to epileptogenesis in patients carrying these mutations (Vitko et al. 2007). Patch clamp recordings of surface-expressed receptors carrying CACNA1A-mutations associated with familiar hemiplegic migraine/epilepsy further supported the hypothesis of a gain-of-function effect by showing that these mutations increase single channel calcium influx (Tottene et al. 2002). Both an increase in channel expression and a larger calcium influx would raise intracellular calcium levels, which subsequently would affect the expression of many different genes via calcium signaling. The hypothesis that an increase in calcium channel function can be a possible pathomechanism underlying absence epilepsy is also supported by mice models overexpressing the Cacna1g gene. These mice developed a pure absence epilepsy phenotype with frequent stereotyped, rhythmic cortical spike-wave discharges and periods of behavioral arrest (Ernst et al. 2009).

Ca 2+

ATP

ROS

Necrosis/apoptosis

Seizures

However, gain-of-function is not the only mechanism that has been described for calcium channel mutations that cause absence epilepsy. The opposite, i.e. a loss-of-function effect, has also been observed. This is best demonstrated by the various spontaneous mouse models that exist for absence epilepsy. These mice are carrying point mutations within the Cacna1a gene that were named tottering, rolling, rocker, leaner, and tg4J (Lorenzon et al. 1998; Miki et al. 2008; Noebels 1986; Xie et al. 2007; Zwingman et al. 2001). Each mutation is thought to have a negative effect on the function of certain conserved domains within the calcium channel. In cases of the tottering and roller mutations, for example, this leads not only to a decrease of the voltage sensitivity of single channel current activation but also reduces membrane expression of the channel in heterologous expression experiments. Both of these mechanisms are likely to significantly decrease but not abolish channel function (Noebels 2012). A complete loss-of-function can be best studied in knockout animal models. Mice without a functional Cacna1a calcium channel gene showed an absence epilepsy-like phenotype with brief episodes of cessation of movement, but were also affected by ataxia that progressively worsened up to the point of premature death (Jun et al. 1999). So far, it remains unclear why experimental data attribute both loss- and gain-of-function effects to mutations that cause absence epilepsy. Part of the answer might be that the models used to study the functional effects of calcium channel mutations are not necessarily always representative of the (patho-) mechanisms taking place in the brain of a patient with absence epilepsy. It has, for example, been shown that, at least for some mutations, opposite effects with regard to receptor surface density and other functional characteristics can be observed when comparing expression of these mutations between HEK (human embryonic kidney) cells and primary cultures of neurons (Tottene et al. 2002). This apparent contradiction makes it difficult to predict the effects that mutations

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have in vivo and even renders it possible that these effects are not the same throughout the brain. Indeed, it is entirely possible that these effects vary between different brain tissues or neuron subpopulations. Furthermore, it cannot be excluded that more than one of the results obtained from model systems are representative of the real situation and that both types of effects, loss and gain, might indeed result in the same clinical outcome. The pathology underlying absence epilepsy and other seizure disorders might not be the same in each patient, and there are most likely many different mechanisms that are able to produce similar clinical phenotypes. Most calcium channel mutations that are associated with epilepsy in humans or in animal models cause a rather benign form of this disorder (the exception being the above-mentioned Cacna1a knock-out mouse). But in humans, at least one mutation is known that gives rise to a much more serious and rather unique neurological phenotype. The mutation, amino acid exchange p.S218L, is located in the CACNA1A gene. It has been found to cause hemiplegic migraine and cerebral edema with coma (and even death) as a delayed reaction following minor head trauma in some patients with this mutation (Chan et al. 2008; Curtain et al. 2006; Debiais et al. 2009; Kors et al. 2001). During childhood, these patients might experience isolated seizures, which are an unusual feature in familial hemiplegic migraine patients (Chan et al. 2008; Debiais et al. 2009). Heterologous expression experiments revealed that the effect the CACNA1A-S218L mutation has on channel function differs from that exhibited by the abovedescribed familial hemiplegic migraine/epilepsy-associated CACNA1A mutations. The CACNA1A-S218L calcium channels open and show significant currents at voltages that are close to the resting potential of many neurons (−60 to −50 mV). This means that channels with this particular mutation are able to open at voltages at which the wildtype channels remain closed. CACNA1A-S218L calcium channels display an unusual small extent of inactivation during long depolarizations and a very fast recovery from inactivation (Tottene et al. 2002). These results, that were obtained with HEK293 cells, were later confirmed in cerebellar granule cells from primary cultures, a model system that, compared to HEK cells, is probably better able to mimic the situation in vivo (Tottene et al. 2002). The unusual functional characteristics demonstrated by CACN A1A-S218L are likely to be responsible for the dramatic clinical features. Minor head traumas are a known trigger for attacks in patients with familial hemiplegic migraine caused by other calcium channel mutations, but in these patients they have never been observed to lead to cerebral edema. In CACNA1A-S218L patients, the combination of a low activation threshold with a slow inactivation is thought to lead to long-lasting cortical spreading depression that is

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significantly prolonged, in comparison to healthy subjects with the same kind of trauma. One of the consequences would be a longer activation of NMDA receptors after minor head trauma in CACNA1A-S218L patients. The result would be intracellular calcium overload which in turn is able to trigger a persistent depolarization of neurons, mitochondrial dysfunction resulting in energy failure, and an increased production of reactive oxygen species (GuemezGamboa et al. 2011; Gursoy-Ozdemir et al. 2004; Heinemann et al. 1999; Vizi et al. 2013). The accumulation of these potentially cytotoxic substances could induce edema, for example, by damaging the blood–brain barrier. It has also been shown that minor head trauma enhances the vulnerability of neurons to excitotoxic insults, thus a longlasting cortical spreading depression might even directly damage neurons (Arundine et al. 2004; Tottene et al. 2002). Interestingly, the knock-in mouse model for CACN A1A-S218L has shown that this mutation renders Purkinje cells hyperexcitable and evokes irregular and slower Purkinje cell firing (Gao et al. 2012). This is most likely an effect due to the abnormal calcium influx caused by the shift of the activation threshold of the CACNA1A channel towards more hyperpolarized levels. The negative shift leads to a potential overlap between the calcium channels activation threshold with the initiation threshold of sodiumdependent action potentials. This in turn could promote somatic burst-firing and irregular activity patterns. Furthermore, the possibility exists that disturbed calcium signaling leading to epilepsy might be caused by mutations that were not inherited through the germline but arise de novo during embryogenesis or later and are therefore only present in certain brain areas (mosaicism). Although not yet systematically studied, there are some examples where mosaicism for a certain mutation was found to be the cause of seizures. An example would be the finding that somatic mosaicism for a mutation within GNAO1, a gene which encodes a Gαo subunit of heterotrimeric G proteins, causes seizures by affecting the inhibition of calcium currents by norepinephrine (Nakamura et al. 2013).

Conclusion The examples summarized here demonstrate that calcium ions are able to cause seizures and other neurological symptoms by many different mechanisms. This astonishing pathogenetic variety reflects the enormous versatility of calcium signaling that allows it to participate in many different physiological and pathological functional pathways. These pathways constitute promising therapeutic targets and a better understanding of their role in epileptogenesis and seizure generation will greatly facilitate the development of new antiepileptic drugs.

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