Canine and Feline Epilepsy: Diagnosis and Management

Preface

Seizures are one of the most common neurological conditions encountered in small animal practice. Epilepsy is the most common chronic neurological disease in dogs; it is often associated with dramatic clinical signs, lifelong treatment and potential effects on the animal quality of life and lifespan. The emotional and financial impact of this disease on the pet-owners can be dramatic.

Diagnosis and management of the seizure patient can be challenging. To date, information on various aspects of this topic has been covered in individual chapters in neurology, internal medicine and pharmacology textbooks and in scientific articles. Therefore consultation of numerous publications has been necessary to obtain comprehensive knowledge.

The authors have compiled this textbook in order to provide information on multiple aspects of canine and feline seizures and epilepsy such as pathophysiology, classification, aetiologies and differential diagnoses, epidemiology, diagnostic investigations and emergency and maintenance treatment. Mechanism of action, metabolism and pharmacokinetics, pharmacokinetic interactions and adverse reactions, dosing, monitoring recommendations and efficacy of old and new generation antiepileptic medications are presented in detail. A glossary on pharmacological terminology has been added at the end of the book to help understanding in this area. Extensive referencing has been provided.

Having all of this information available in one textbook should help to improve knowledge on this complex subject and subsequently help veterinarians to improve the care of dogs and cats with seizure activity.

The authors are very grateful to the invited authors, Holger Volk and Fiona James, for their invaluable contribution in their respective area of expertise, Pathophysiology of refractory seizures (Chapter 2) and Electroencephalography (Chapter 11), and to colleagues who have contributed images. We also wish to thank the publisher CABI and particularly our senior editorial assistant, Alexandra Lainsbury.

Due to production requirements the number of colour images is greater in the e-book than in the printed book. The layout of colour images in the printed book has been determined by the publisher’s production.

We hope that this textbook provides useful information for veterinary students, veterinary general practitioners, as well as veterinary interns, residents and specialists in neurology or in disciplines related to neurology (e.g. internal medicine, oncology, surgery, behavioural medicine and pharmacology). The depth of the information provided will allow those who would like a little bit more detail in certain areas hopefully to find this; however, we hope that the book also serves as a practical source for advancing the treatment of the most routine and most challenging seizure cases.

The information provided in this textbook is up to date to the best of the authors’ knowledge at the time of production. The field of veterinary science is rapidly evolving and advances in diagnosis and treatment are likely to occur in the next years. Therefore knowledge would require constant updating to provide optimal management of our patients.

Luisa De Risio

Simon Platt

October 2013

1 Pathophysiology of Seizure Activity

Simon Platt BVM&S MRCVS Dipl. ACVIM (Neurology) Dipl. ECVN

Professor Neurology and Neurosurgery Service, Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA

Several decades have been devoted to the study of the pathophysiology of epilepsy. Increasing knowledge in the field has only contributed to a partial understanding of the underlying mechanisms. Nevertheless, insight into the pathophysiology of epilepsy and its underlying histological and neurochemical alterations has contributed to rational development strategies of new anti-epileptic medications (AEMs). Although various epileptic syndromes in people have been shown to differ pathophysiologically, they apparently share common ictogenesis-related characteristics such as increased neuronal excitability and synchronicity. Emerging insights point to alterations of synaptic functions and intrinsic properties of neurons as common mechanisms underlying hyperexcitability. Progress in the field of molecular genetics has revealed arguments in favour of this hypothesis as mutations of genes encoding ion channels were recently discovered in some forms of human epilepsy.

Epileptic seizures arise from an excessively synchronous and sustained discharge of a group of neurons. The single feature of all epileptic syndromes is a persistent increase of neuronal excitability. Abnormal cellular discharges may be associated with a variety of causative factors such as trauma, oxygen deprivation, tumours, infection and metabolic derangements. However, no specific causative factors are found in many dogs and cats suffering from epilepsy.

Underlying causes and pathophysio-logical mechanisms are (partially) understood for some forms of epilepsy, at least in people, e.g. epilepsies caused by disorders of neuronal migration and monogenic epilepsies. For several other types of epilepsy, current knowledge is only fragmentary. This chapter will review several areas that are understood to contribute to the evolution and maintenance of epilepsy. The genetics of epilepsy are discussed in Chapter 6.

The Electrical Basis of Nerve Cell Function

At the most fundamental level, the nervous system is a function of its ionic milieu, the chemical and electrical gradients that create the setting for electrical activity. Therefore, some of the most easily appreciated controls on excitability are the ways the nervous system maintains the ionic environment. An example is the electrical basis of resting membrane potential. Resting potential is set normally so that neurons are not constantly firing but are close enough to threshold so that it is still possible that they can discharge, given that action potential generation is essential to CNS function. The control of resting potential becomes critical to prevent excessive discharge that is typically associated with seizures.

Normally a high concentration of potassium exists inside a neuron and there is a high extracellular sodium concentration, as well as additional ions, leading to a net transmembrane potential of −60 mV (Scharfman, 2007). If the balance is perturbed (e.g. if potassium is elevated in the extracellular space), this can lead to depolarization that promotes abnormal activity in many ways (Somjen, 2002): terminals may depolarize, leading to transmitter release, and neurons may depolarize, leading to action potential discharge. Pumps are present in the plasma membrane to maintain the chemical and electrical gradients, such as the sodium–potassium ATPase, raising the possibility that an abnormality in these pumps could facilitate seizures. Indeed, blockade of the sodium–potassium ATPase can lead to seizure activity in experimental preparations (Vaillend et al., 2002), suggesting a role in epilepsy (Grisar et al., 1992). The sodium–potassium pump is very interesting because it does not develop in the rodent until several days after birth, and this may contribute to the greater risk of seizures in early life (Haglund et al., 1985; Fukuda and Prince, 1992). In addition to pumps, glia also provide important controls on extracellular ion concentration, which has led many to believe that glia are just as important as neurons in the regulation of seizure activity (Duffy and MacVicar, 1999; Fellin and Haydon, 2005). Thus, the control of the ionic environment provides many potential targets for novel anticonvulsants. It is important to bear in mind that seizures, by themselves, can lead to the changes in the transmembrane gradients. For example, seizures are followed by a rise in extracellular potassium, a result of excess discharge. This can lead to a transient elevation in extracellular potassium that can further depolarize neurons. Thus, the transmembrane potential is a control point that, if perturbed, could elicit seizures and begin a ‘vicious’ cycle, presumably controlled by many factors that maintain homeostasis, such as pumps and glia.

The ionic basis of the action potential is another example of a fundamental aspect of neurobiology that can suggest potential mechanisms of seizures. Neurons are designed to discharge because of an elegant orchestration of sodium and potassium channels that rely on chemical and ionic gradients across the cell membrane. Abnormalities in the sodium channel might lead to a decrease in the threshold for an action potential to occur if the method by which sodium channel activation is controlled alters in any way (i.e. sodium channels are activated at more negative resting potentials or sodium channel inactivation is impaired). Indeed, it has been shown that mutations in the subunits of the voltage-dependent sodium channels can lead to epilepsy. A specific syndrome, generalized epilepsy with febrile seizures, is caused by mutations in selected genes responsible for subunits of the voltage-dependent sodium channel (Meisler et al., 2001). The mutation does not block sodium channels, presumably because such a mutation would be lethal, but they modulate sodium channel function. This concept, that modulation, rather than essential function, is responsible for genetic epilepsies, has led to a greater interest in directing the development of new anticonvulsants at targets that are not essential to, but simply influence, CNS function.

Synaptic Transmission

Research into seizures has gravitated to mechanisms associated with synaptic transmission, because of its critical role in maintaining the balance between excitation and inhibition. As more research has identified the molecular mechanisms of synaptic transmission, it has become appreciated that defects in almost every step can lead to seizures. Glutamatergic and γ-aminobutyric acid (GABA)-ergic transmission, as the major excitatory and inhibitory transmitters of the nervous system, respectively, have been examined in great detail. It is important to point out, however, that both glutamate and GABA may not have a simple, direct relationship to seizures. One reason is that desensitization of glutamate and GABA receptors can reduce effects, depending on the time-course of exposure. In addition, there are other reasons. GABA-ergic transmission can lead to depolarization rather than hyperpolarization if the gradients responsible for ion flow through GABA receptors are altered. For example, chloride is the major ion that carries current through GABAA receptors, and it usually hyperpolarizes neurons because chloride flows into the cell from the extracellular space. However, the K+Cl− co-transporters (KCCs) that are pivotal to the chloride gradient are not constant. In development, transporter expression changes, and this has led to evidence that one of the transporters, NKCC1, may explain seizure susceptibility early in life (Dzhala et al., 2005). The relationship of glutamate to excitation may not always be simple either. One reason is that glutamatergic synapses innervate both glutamatergic neurons and GABA-ergic neurons in many neuronal systems. Exposure to glutamate could have little net effect as a result, or glutamate may paradoxically increase inhibition of principal cells because the GABA-ergic neurons typically require less depolarization by glutamate to reach threshold. It is surprisingly difficult to predict how glutamatergic or GABA-ergic modulation will influence seizure generation in vivo, given these basic characteristics of glutamatergic and GABA-ergic transmission.

Synchronization

Excessive discharge alone does not necessarily cause a seizure. Synchronization of a network of neurons is involved. Therefore, how synchronization occurs becomes important to consider. There are many ways neurons can synchronize. In 1964, Matsumoto and Ajmone-Marsan found that the electrographic events recorded at the cortical surface during seizures corresponded to paroxysmal depolarization shifts (PDS) of cortical pyramidal cells occurring synchronously (Matsumoto and Marsan, 1964). These studies led to efforts to understand how neurons begin to fire in concert when normally they do not. Glutamatergic interconnections are one example of a mechanism that can lead to synchronization. Indeed, studies of the PDS suggested that the underlying mechanism was a ‘giant’ excitatory postsynaptic potential, although it was debated widely at that time if this was the only cause (Johnston and Brown, 1984). Thus, pyramidal cells of cortex are richly interconnected to one another by glutamatergic synapses. Gap junctions on cortical neurons are another mechanism for synchronization. Gap junctions allow a low-resistance pathway of current flow from one cell to another, so that coupled neurons are rapidly and effectively synchronized. It was thought that gap junctions were rare, so it was unlikely that they could play a major role, but further study led to the appreciation that even a few gap junctions may have a large impact on network function (Traub et al., 2004). Another mechanism of synchronization involves, paradoxically, inhibition.

Many GABA-ergic neurons that innervate cortical pyramidal cells, such as the cell type that controls somatic inhibition (the basket cell), make numerous connections to pyramidal cells in a local area. Therefore, discharge of a single interneuron can synchronously hyperpolarize a population of pyramidal cells. As GABA-ergic inhibition wanes, voltage-dependent currents of pyramidal cells become activated. These currents, such as T-type calcium channels and others, are relatively inactive at resting potential, but hyperpolarization relieves this inhibition. The result is a depolarization that is synchronous in a group of pyramidal cells (Scharfman, 2007).

Some of the changes that develop within the brain of individuals with epilepsy also promote synchronization. Such changes are of interest in themselves because they may be one of the reasons why the seizures are recurrent. These changes include growth of axon collaterals of excitatory neurons, typically those that use glutamate as a neurotransmitter and are principal cells. An example is the dentate gyrus granule cell of hippocampus. In animal models of epilepsy and in patients with intractable temporal lobe epilepsy (TLE), the axons of the granule cells develop new collaterals and the new collaterals extend for some distance. They do not necessarily terminate in the normal location but in a novel lamina, one that contains numerous granule cell dendrites. Electron microscopy has shown that the new collaterals innervate granule cell dendrites, potentially increasing recurrent excitatory circuits. Some argue that recurrent inhibition increases as well as recurrent excitation, but the fact remains that new synaptic excitatory circuits develop that are sparse or absent in the normal brain (Nadler, 2003; Sloviter et al., 2006). The resultant ‘synaptic reorganization’ not only can support synchronization, potentially, but it also illustrates how the plasticity of the nervous system may contribute to epileptogenesis.

Kindling and Epileptogenesis

Goddard (1967) was the first to describe that periodic stimulation of neural pathways progressively leads to recurrent behavioural and electrographic seizures. Kindling procedures have provided a substrate for the study of the role of enhanced synaptic efficacy in seizure disorders. It is now considered to be a first choice experimental procedure in the study of the potential mechanisms of epileptogenesis. The phenomenon can be evoked in various brain regions, but amygdala kindling is most frequently used in epilepsy research as a model for complex focal (partial) seizures (Fisher, 1989). Although kindling has been shown to be phenomenologically different from other types of plastic changes in the central nervous system, there are many points of similarity between kindling and the process of long-term potentiation (Sutula et al., 1989).

Kindling has been shown to depend upon functional as well as structural changes in glutamatergic synapses. The anticonvulsant effects of glutamate receptor blocking agents like N-methyl-D-aspartate (NMDA) antagonists seem to be at least partly due to their inhibitory effects on in vitro kindling.

Ictogenesis

Excitability is a key feature of ictogenesis that may originate from individual neurons, neuronal environment or a population of neurons. Excitability arising from single neurons may be caused by alterations in membrane or metabolic properties of individual neurons (Traub et al., 1996). When regulation of environmental, extracellular concentrations of ions or neurotransmitters is suboptimal, the resulting imbalance might enhance neuronal excitation. Collective anatomic or physiologic neuronal alterations may convert neurons into a hyper-excitable neuronal population. In reality, these three theoretical mechanisms are thought to interact during specific ictal episodes. Each epileptic focus is unique as the differential contribution of these three concepts leading to ictal events is thought to differ from focus to focus.

Excitability arising from individual neurons

Functional and perhaps structural changes occur in the postsynaptic membrane, thus altering the character of receptor protein-conductance channels, thereby favouring development of paroxysmal depolarizing shifts (PDS) and enhanced excitability. Epileptic neurons appear to have increased Ca²+ conductance. It may be that latent Ca²+ channels are used, that the efficacy of Ca²+ channels is increased or that the number of Ca²+ channels is chronically elevated. However, development of burst activity depends on the net inward current and not on the absolute magnitude of the inward current. When extracellular K+ concentrations are increased (as during seizure activity), the K+ equilibrium across the neuronal membrane is reduced, resulting in reduced outward K+ currents. The net current will become inward, depolarizing the neuron to the extent that Ca²+ currents will be triggered. This results in a PDS and a burst of spikes (Dichter and Ayala, 1987).

Excitability arising from neuronal microenvironment

Both functional and structural alterations occur in epileptic foci. The functional changes involve concentrations of cations and anions, metabolic alterations, and changes in neuro-transmitter levels. The structural changes involve both neurons and glia. Excessive extracellular K+ depolarizes neurons and leads to spike discharge. During seizures, changes in extracellular Ca²+ (a decrease of 85%) precede those of K+ by milliseconds and Ca²+ levels return to normal more quickly than K+. Glia are able to clear neurotransmitters from the extracellular space and to buffer K+, thus correcting the increased extracellular K+ concentrations that occur during seizures. Epileptic foci may show proliferation of glia that differ however in morphological and physiological properties (Bordey and Sontheimer, 1998). Gliosis will affect glial K+ buffering capacity and hence may contribute to seizure generation.

The epileptic cell population

Collective anatomic or physiologic neuronal alterations might produce progressive, network-dependent facilitation of excitability, perhaps coupled with a decrease of inhibitory influences, e.g. due to selective loss of inhibitory neurons. Mossy fibre sprouting (MFS) is an example of neuronal alterations leading to increased excitability (Cavazos et al., 1991). MFS was demonstrated in patients with refractory temporal lobe epilepsy with hippocampal sclerosis on neuroimaging as well as in numerous animal models of temporal lobe epilepsy (Sutula et al., 1988, 1989). In normal conditions, the dentate granule cells limit seizure propagation through the hippocampal network. However, the formation of recurrent excitatory synapses between dentate granule cells, as is thought to occur after MFS, may transform the dentate granule cells into an epileptogenic population of neurons (McNamara, 1999). Possibly, a vicious cycle develops: seizures cause neuronal death, which results in MFS, which in turn increases seizure frequency.

Mechanisms of Interictal–Ictal Transition

Mechanisms producing signal amplification, synchronicity and spread of activity are likely to be involved in interictal–ictal transitions. In vivo, interictal–ictal transition can seldom be attributed to one theoretical mechanism, but often results from the interaction of different mechanisms.

Nonsynaptic mechanisms

Alterations in ionic microenvironment

Repetitive ictal and interictal activity causes increases in extracellular K+ leading to increased neuronal excitability (Moody et al., 1974). Some neurons are very sensitive to changes in membrane K+ currents, e.g. pyramidal cells in the CA1 region of the hippo-campus (Rutecki et al., 1985).

Active ion transport

Activation of the Na+–K+ pump is important for regulation of neuronal excitability during excessive neuronal discharges. Substances like ouabain that block the Na+–K+ pump can induce epileptogenesis in animal models. Hypoxia or ischaemia can result in Na+–K+ pump failure thus promoting interictal–ictal transition. A Cl−–K+ co-transport mechanism controls the intracellular Cl− concentration and the Cl− gradient across the cell membrane, which regulates effectiveness of GABA-activated inhibitory Cl− currents. Interference with this process could cause a progressive decrease in the effectiveness of GABA-ergic inhibition leading to increased excitability (Engelborghs et al., 2000).

Presynaptic terminal bursting

The amount of transmitter released is related to depolarization of presynaptic terminals. Changes in axon terminal excitability will have effects on synaptic excitation (Engelborghs et al., 2000). Abnormal bursts of action potentials occur in the axonal arborizations of thalamocortical relay cells during epileptogenesis. Since one thalamocortical relay cell ends on a large number of cortical neurons, synchronization can occur, which might play an important role in interictal–ictal transition (Engelborghs et al., 1998a).

Ephaptic interaction

Ephaptic interactions are produced when currents from activated neurons excite adjacent neurons in the absence of synaptic connections. Ephaptic effects are strongly dependent on the size of the extracellular space. When extracellular space is small, ephaptic interactions are promoted (Traub et al., 1985).

Synaptic mechanisms

Two theoretical mechanisms can occur: decreased effectiveness of inhibitory synaptic mechanisms or facilitation of excitatory synaptic events. Both mechanisms will be discussed below.

Neurochemical Mechanisms Underlying Epilepsy

GABA

The GABA hypothesis of epilepsy implies that a reduction of GABA-ergic inhibition results in epilepsy whereas an enhancement of GABA-ergic inhibition results in an anti-epileptic effect (Wong and Watkins, 1982; De Deyn and Macdonald, 1990; De Deyn et al., 1990). Inhibitory postsynaptic potentials (IPSPs) gradually decrease in amplitude during repetitive activation of cortical circuits. This phenomenon might be caused by decreases in GABA release from terminals, desensitization of GABA receptors that are coupled to increases in Cl− conductance or alterations in the ionic gradient because of intracellular accumulation of Cl− (Wong and Watkins, 1982). In case of intracellular accumulation of Cl−, passive redistribution is ineffective. Moreover, Cl−–K+ co-transport becomes less effective during seizures as it depends on the K+ gradient. As Cl−–K+ co-transport depends on metabolic processes, its effectiveness may be affected by hypoxia or ischaemia as well. These mechanisms may play a critical role in ictogenesis and interictal–ictal transition. Several studies have shown that GABA is involved in pathophysiology of epilepsy in both animal models and patients suffering from epilepsy. GABA levels and glutamic acid decarboxylase (GAD) activity were shown to be reduced in epileptic foci surgically excised from patients with intractable epilepsy and in CSF of patients with certain types of epilepsy (De Deyn et al., 1990).

A reduction of 3H–GABA binding has been reported in human brain tissue from epileptic patients whereas PET studies demonstrated reduced benzodiazepine receptor binding in human epileptic foci (Savic et al., 1996). The degree of benzodiazepine receptor reduction showed a positive correlation with seizure frequency. The GABA receptor complex is involved in various animal models of epilepsy as well. Low CSF levels of GABA were revealed in dogs with epilepsy (Loscher and Schwartz-Porsche, 1986). Reduced GAD levels were revealed in the substantia nigra of amygdala-kindled rats (Loscher and Schwark, 1985). Significant alterations in GABA and benzodiazepine binding have been shown in the substantia nigra of genetically seizure-prone gerbils (Olsen et al., 1985). Mice with a genetic susceptibility to audiogenic seizures have a lower number of GABA receptors than animals of the same strain that are not seizure prone (Horton et al., 1982). Several endogenous (guanidino compounds) and exogenous (e.g. bicuculline, picrotoxin, penicillin, pilocarpine, pentylenetetrazol) convulsants inhibit GABA-ergic transmission through inhibition of GABA synthesis or through interaction with distinct sites at the postsynaptic GABAA receptor (De Deyn and Macdonald, 1990; D’Hooge et al., 1996). Convulsant agents that block synaptic GABA-mediated inhibition, amplify the dendritic spike-generating mechanism that involves Ca²+ (Dichter and Ayala, 1987; Fisher, 1989). Synaptic inputs are thought to trigger and synchronize this process throughout a population of cells, which then might result in an epileptic seizure. Several AEMs are GABA analogues, block GABA metabolism or facilitate postsynaptic effects of GABA. However, a study evaluating dose-dependent behavioural effects of single doses of vigabatrin in audiogenic sensitive rats, suggests that the anti-epileptic properties of vigabatrin not only depend on GABA-ergic neurotransmission but might also be explained by decreased central nervous system levels of excitatory amino acids or increased glycine concentrations (Engelborghs et al., 1998b).

Glutamate

In rodent models, altering glutamate receptor or glutamate transporter expression by knockout or knockdown procedures can induce or suppress epileptic seizures (Chapman et al., 1996; Kabova et al., 1999; Chapman, 2000). Regardless of the primary cause, synaptically released glutamate acting on ionotropic and metabotropic receptors appears to play a major role in the initiation and spread of seizure activity (Meldrum, 1994; Chapman et al., 1996; Chapman, 2000). Glutamatergic synapses play a critical role in all epileptic phenomena. Activation of both ionotropic and metabotropic postsynaptic glutamate receptors is proconvulsant. Antagonists of N-methyl-D-aspartate (NMDA) receptors are powerful anticonvulsants in many animal models of epilepsy. Several genetic alterations have been shown to be epileptogenic in animal models.

Glutamate receptors

Studies of epileptiform discharges in hippocampal slices show that the characteristic burst discharge, associated with a ‘paroxysmal depolarizing shift’, is dependent on activation of AMPA receptors for its initial components and NMDA receptors for the later elements (Bengzon et al., 1999; Mazarati and Wasterlain, 1999; Meldrum et al., 1999).

AMPA

AMPA receptor antagonists, either competitive or non-competitive, are anticonvulsant in rodent models (Rogawski and Donevan, 1999). Thus, altered function of AMPA receptors could contribute to proconvulsant or anticonvulsant effects (Meldrum et al., 1999). Evidence has accumulated that Ca²+-permeable AMPA receptors may play a role in epileptogenesis and the brain damage occurring during the prolonged seizures (Rogawski and Donevan, 1999). Because Ca²+-permeable AMPA receptors are predominantly expressed in GABA-ergic inter-neurons, it is hypothesized that some forms of epilepsy might be caused by reduced GABA inhibition resulting from Ca²+-permeable AMPA receptor-mediated excitotoxic death of interneurons (Rogawski and Donevan, 1999).

NMDA

NMDA receptor antagonists are potent anticonvulsants in many animal models, suggesting a role for these receptors in epileptogenesis (Patrylo et al., 1999). It is known that enhancing NMDA receptor-mediated excitatory actions (e.g. by lowering extracellular Mg) produces epileptiform activity in experimental models of ‘kindled’ epilepsy (Chapman, 1998, 2000). It has been postulated that NMDA receptors may change after neuronal damage (Rice and DeLorenzo, 1998). New receptors are formed that have either less sensitivity to ambient Mg or more sensitivity to ambient glycine; increased excitability could occur within local circuits where the circuitry itself is not altered (or may occur in addition to circuit alterations) (Meldrum et al., 1999). As it is known that the NMDA receptor is subject to modulation by a variety of endogenous agents, including glycine (as a co-agonist with glutamate), polyamines, steroids, neuropeptides (Vezzani et al., 2000b), pH, the redox state of the receptor, and NO, there are many chronic alterations in NMDA receptors that could underlie long-term changes in excitability and, thereby, epilepsy. Presently, there are no data to support changes in any of these regulatory factors in chronic epilepsy, but it is distinctly possible that alterations in one or more of these will be shown to be responsible for one or another form of inherited epilepsy.

Kindling is the most extensively studied animal model of epileptogenesis, and this has demonstrated the unique importance of NMDA receptors in the creation of seizure activity (Bengzon et al., 1999; Meldrum et al., 1999). In kindling, repeated electrical stimuli in the limbic system lead to a progressive increase of seizure susceptibility. When the animal responds to stimuli with generalized convulsions, it has developed a permanent epileptic condition. Activation of NMDA receptors and levels of NMDA receptor function are critical in kindling epilepsy (Bengzon et al., 1999). Selective NMDA-receptor antagonists retard kindling development and can also, at higher doses, have an anticonvulsant effect (Bengzon et al., 1999; Trist, 2000).

Metabotropic receptors

On account of these receptors’ responsibility for regulating glutamatergic and GABA-ergic neurotransmission, it is not surprising that mGluRs strongly influence the induction, propagation and termination of epileptic activity in the central nervous system (CNS) (Doherty and Dingledine, 2002). Pharmacological studies with mGluR group specific agonists and antagonists provide a relatively clear picture for Group I, with agonists being convulsant and antagonists being anticonvulsant (Meldrum et al., 1999; Doherty and Dingledine, 2002; Sayin and Rutecki, 2003). The picture is more complicated for the Group II and III receptors but anticonvulsant effects have been described for agonists of both these groups (Meldrum et al., 1999).

Glutamate transporters

In addition to receptor abnormalities, glutamate transporters, responsible for the removal of glutamate from the extracellular fluid, have been implicated in epilepsy (Meldrum et al., 1999). In situ hybridization studies have shown that the mRNA responsible for the rat glial glutamate transporter (GLT) is reduced in several brain regions in epilepsy-prone rats (Meldrum et al., 1999). GLT ‘knockout’ mice have been bred to provide homozygous mice, in which the GLT protein is not detected. In such mutant mice, glutamate uptake in cortical synaptosomes is 5.8% compared with the wild-type (Meldrum et al., 1999). The mutant mice show spontaneous seizures, with wild running and tonic extension, which is frequently fatal. In chronic seizure models (kindled seizures, spontaneous seizures and genetically epilepsy-prone rats), there are numerous reports of increases in extracellular glutamate during seizures (Meldrum et al., 1999). This strongly suggests that in these chronic models there are sustained functional alterations in mechanisms relating to the synaptic release of glutamate or its transport. GLT-1 astrocytic expression was reduced in four Shetland sheepdogs with IE (Morita et al., 2005). In these dogs it was suggested that decreased expression of the transporter might be related to development of status epilepticus.

There is not as yet any genetically determined epilepsy syndrome occurring spontaneously in man or mouse that can be ascribed to a primary gene defect involving a glutamate receptor or transporter.

Targets for treatment

In animal models of epilepsy, antagonists acting at NMDA receptors, AMPA receptors or at Group I metabotropic receptors have potent anticonvulsant actions (Meldrum and Chapman, 1999; Rogawski and Donevan, 1999; Chapman, 2000; Moldrich et al., 2003).

NMDA receptor antagonists have been successful in stopping the maintenance phase of self-sustaining status epilepticus (SE) in rats, which suggests that these compounds may have a promising role in the treatment of unrelenting seizure activity such as SE (Mazarati and Wasterlain, 1999). Studies with selective AMPA receptor antagonists have indicated that AMPA receptors are potentially promising anticonvulsant drug targets, but at present this is uncertain (Rogawski and Donevan, 1999).

In genetic mouse models, mGlu1/5 antagonists and mGlu2/3 agonists are effective against absence seizures. Thus, antagonists at Group I mGlu receptors and agonists at Groups II and III mGlu receptors are potential anti-epileptic agents, but their clinical usefulness will depend on their acute and chronic side-effects (Moldrich et al., 2003). Potential also exists for combining mGlu receptor ligands with other glutamatergic and non-glutamatergic agents to produce an enhanced anticonvulsant effect (Moldrich et al., 2003).

The Veterinary Perspective

Idiopathic epilepsy (see Chapter 6) is the most common cause of seizures in dogs (Podell and Hadjiconstantinou, 1997). Low levels of GABA and high levels of glutamate have been detected in the cerebrospinal fluid of epileptic dogs independent of time relation to recent seizure activity (Podell and Hadjiconstantinou, 1997). The glutamate elevations are not related whether the seizures were focal or generalized in character (Podell and Hadjiconstantinou, 1997). These findings may indicate the brains of epileptic dogs are under a state of chronic over-excitation. Although a separate study found that lower CSF GABA concentration was associated with a reduced response to phenobarbital therapy in dogs, there was no association between CSF glutamate and response to this therapy (Podell and Hadjiconstantinou, 1999). However, a negative association was found between CSF glutamate:GABA ratio and response to phenobarbital therapy (Podell and Hadjiconstantinou, 1999). Therefore glutamate-mediated mechanisms may be useful targets for anticonvulsant therapy in dogs. Intracerebral microdialysis was used to demonstrate elevation of extracellular levels of glutamate in four Shetland sheep-dogs with IE, suggesting an important role in the occurrence of seizure activity (Morita et al., 2005).

Gabapentin (see Chapter 17), a relatively new human anticonvulsant, has been evaluated in dogs refractory to phenobarbitone and potassium bromide with an approximate 50% success rate.

Gabapentin has been shown to modestly decrease glutamate levels in the brain (Errante and Petroff, 2003). Another new anticonvulsant, topiramate (see Chapter 19), produces its antiepileptic effect by several mechanisms, one of which is inhibition of kainite-mediated glutamate receptors (Angehagen et al., 2003a). This drug has also been demonstrated to protect neurons from excitotoxic levels of glutamate, potentially preventing brain damage during seizure activity (Angehagen et al., 2003b).

Catecholamines

Abnormalities of CNS catecholamines have been reported in several genetic models of epilepsy. In the spontaneous epileptic rat, dopamine was decreased in the caudate nucleus whereas noradrenaline was increased in the midbrain and brainstem (Hara et al., 1993). Decreased levels of dopamine have been found in epileptic foci of epilepsy patients (Mori et al., 1987). In animal models of absence epilepsy, seizures are exacerbated by dopamine antagonists while alleviated by dopamine agonists (Snead, 1995). These results suggest that decreased dopamine facilitates appearance of seizures by lowering the threshold triggering such seizures. Tottering mice have an absence-like syndrome that is characterized by episodes of behavioural arrest associated with 6 to 7 Hz cortical SW EEG discharges. Selective destruction of the ascending noradrenergic system at birth prevents the onset of the syndrome. Therefore, it has been suggested that the syndrome is caused by a noradrenergic hyperinnervation of the forebrain (Engelborghs et al., 2000). Recent data indicate that the serotonergic system regulates epileptiform activity in a genetic rat model of absence epilepsy as intraperitoneal or intracerebroventricular administration of 8- OHDPAT caused marked and dose-dependent increases in number and duration of discharges (Gerber et al., 1998).

Opioid peptides

In experimental studies, opioids and opioid peptides had both convulsant and anticonvulsant properties (Engelborghs et al., 2000). Kappa agonists suppress electrical discharges in an animal model of absence epilepsy (Przewlocka et al., 1995). Peptides with a μ-agonist action induce hippocampal or limbic seizures when administered intraventricularly possibly due to inhibition of inhibiting interneurons. In patients with complex partial seizures, PET studies pointed out that μ-receptor density is increased in the temporal cortex (Mayberg et al., 1991).

Inflammatory Mechanisms Underlying Epilepsy

Over the past 10 years an increasing body of clinical and experimental evidence has provided strong support to the hypothesis that inflammatory processes within the brain might constitute a common and crucial mechanism in the pathophysiology of seizures and epilepsy (Vezzani et al., 2011). The first insights into the potential role of inflammation in human epilepsy were derived from clinical evidence indicating that steroids and other anti-inflammatory treatments displayed anticonvulsant activity in some drug-resistant epilepsies (Wirrell et al., 2005; Wheless et al., 2007). Additional evidence came from febrile seizures in people, which always coincide with, and are often caused by, a rise in the levels of pro-inflammatory agents (Dube et al., 2007). Evidence of immune system activation in some patients with seizure disorders, the high incidence of seizures in autoimmune diseases, and the discovery of limbic encephalitis as a cause of epilepsy led to the suggestion that immune and inflammatory mechanisms have roles in some forms of epilepsy (Aarli, 2000; Bien et al., 2007; Vincent and Bien, 2008; Vezzani et al., 2011).

Evidence is emerging that inflammation might be a consequence as well as a cause of epilepsy. Several inflammatory mediators have been detected in surgically resected brain tissue from human patients with refractory epilepsies, including temporal lobe epilepsy (TLE) and cortical dysplasia-related epilepsy (Choi et al., 2009; Vezzani et al., 2011). The finding that brain inflammation occurred in epilepsies that were not classically linked to immunological dysfunction highlighted the possibility that chronic inflammation might be intrinsic to some epilepsies, irrespective of the initial insult or cause, rather than being only a consequence of a specific underlying inflammatory or autoimmune aetiology. The mounting evidence for a role for inflammatory processes in human epilepsy has led to the use of experimental rodent models to identify putative triggers of brain inflammation in epilepsy, and to provide mechanistic insights into the reciprocal causal links between inflammation and seizures (Vezzani et al., 2011). Experimental studies have shown that seizure activity per se can induce brain inflammation, and that recurrent seizures perpetuate chronic inflammation. Seizure-associated cell loss can contribute to inflammation but is not a prerequisite for inflammation to occur. In addition, models of systemic or CNS infections suggested that pre-existing brain inflammation increases the predisposition to seizures, associated with alterations in neuronal excitability and enhanced seizure-induced neuropathology. Additional mechanistic insights into the role of inflammation in seizures and the development of epilepsy have been gained through use of pharmacological approaches that interfere with specific inflammatory mediators and from changes in seizure susceptibility in genetically modified mice with perturbed inflammatory pathways (Campbell et al., 1993; Kelley et al., 1999; Vezzani et al., 2000a; Balosso et al., 2005).

Inflammation consists of the production of a cascade of inflammatory mediators (a dynamic process), as well as anti-inflammatory molecules and other molecules induced to resolve inflammation, as a response to noxious stimuli (such as infection or injury), or immune stimulation, and is designed to defend the host against pathogenic threats. Inflammation is characterized by the production of an array of inflammatory mediators from tissue-resident or blood-circulating immuno-competent cells, and involves activation of innate and adaptive immunity. Both innate and adaptive immunity have been implicated in epilepsy, and microglia, astrocytes and neurons are believed to contribute to the innate immunity-type processes that cause inflammation of the brain. The brain has traditionally been considered an immunoprivileged site because of the presence of the blood–brain barrier (BBB), the lack of a conventional lymphatic system, and the limited trafficking of peripheral immune cells. Nevertheless, both the innate and adaptive immune responses are readily evoked within the CNS in response to pathogens, self-antigens, or tissue injury of several aetiologies. Microglia, astrocytes, neurons, BBB endothelial cells, and peripheral immune cells extravasating into brain parenchyma can all produce proinflammatory and anti- inflammatory molecules (Ransohoff et al., 2003; Banks and Erickson, 2010).

The contribution of each cell population to brain inflammation depends on the origin (for example, CNS versus systemic) and the type (for example, infectious versus sterile) of the initial precipitating event (Glass et al., 2010). The BBB represents a key regulatory element of the communication between intrinsic brain cells and peripheral immunocompe-tent cells. As noted above, an inflammatory response in the CNS can be induced in the absence of infection. Brain inflammation has been reported following ischaemic stroke or traumatic brain injury (TBI), and during chronic neurodegenerative diseases. In all these conditions, pronounced activation of microglia and astrocytes takes place in brain regions affected by the specific disease, and these cells act as major sources of inflammatory mediators. Recruitment of peripheral immune cells might also occur (Nguyen et al., 2002; Glass et al., 2010). The activation of innate immunity and the transition to adaptive immunity are mediated by a large variety of inflammatory mediators, among which cytokines, polypeptides that act as soluble mediators of inflammation, have a pivotal role (Akira et al., 2001; Nguyen et al., 2002).

These molecules include interleukins (ILs), interferons (IFNs), tumour necrosis factors (TNFs) and growth factors (for example, transforming growth factor (TGF)-β). Cytokines are released by immunocompetent and endothelial cells, as well as by glia and neurons in the CNS, thereby enabling communication between effector and target cells during an immune challenge or tissue injury. Following their release, cytokines interact with one or more cognate receptors. The most extensively studied prototypical inflammatory cytokines in the CNS are IL-1β, TNF and IL-6 (Allan and Rothwell, 2001; Bartfai et al., 2007). Cytokine activity can be regulated at multiple levels, including gene transcription, cleavage of cytokine precursors (for example, pro-IL-1β, pro-TNF) by specific proteolytic enzymes, and cellular release, as well as through receptor signalling (discussed below). All cell types in the brain seem capable of expressing cytokines and their receptors, with low basal expression of these molecules being rapidly up-regulated following CNS insults. Chemokines comprise a specific class of cytokines that act as chemoattractants to guide the migration of leukocytes from blood through the endothelial barrier into sites of infection or injury (Wilson et al., 2010). These cytokines also regulate microglial motility and neural stem cell migration, provide axon guidance during brain development, and promote angiogenesis, neurogenesis and synaptogenesis (Szekanecz and Koch, 2001; Semple et al., 2010). The release of chemokines is often stimulated by proinflammatory cytokines such as IL-1β.

Several mechanisms have been identified that attenuate the inflammatory response, indicating the importance of such strict control for homeostasis and prevention of injury. Regulatory mechanisms include production of proteins that compete with cytokines to bind their receptors, such as IL-1 receptor antagonist protein (IL-1ra), and decoy receptors that bind cytokines and chemokines but are incapable of signalling, thereby acting as molecular traps to prevent such ligands from interacting with biologically active receptors (Mantovani et al., 2001; Dinarello, 2009). Proteins that inhibit cytokine-induced signal transduction (for example, suppressor of cytokine signalling proteins) or transcription (for example, Nurr1-CoREST or activity transcription factor 3), as well as an array of soluble mediators with anti-inflammatory activities (such as IL-10 and TGF-β), are produced concomitantly with proinflammatory molecules to resolve inflammation (Blobe et al., 2000; Khuu et al., 2007; Baker et al., 2009). For example, glucocorticoids, via activation of glucocorticoid receptors and, consequently, down-regulation of nuclear factor-κB (NFκB) and activator protein 1 activity, inhibit innate immune responses and, hence, act as an endogenous anti-inflammatory feedback system. Proinflammatory cytokines are powerful enhancers of glucocorticoid levels in adrenal glands via corticotropin-releasing hormone and adrenocorticotropic hormone (ACTH). Glucocorticoids also elicit immunosuppressive effects through inhibition of leukocyte extravasation from the vasculature, and through regulation of T helper cell differentiation (Sapolsky et al., 1987; Elenkov et al., 1999). The CNS can also negatively regulate the inflammatory response in a reflexive manner, using the efferent activity of the vagus nerve to inhibit release of proinflammatory molecules from tissue macrophages (Vezzani et al., 2000a, 2011; Tracey, 2002).

Do seizures cause inflammation?

In adult rats and mice, induction of recurrent short seizures or single prolonged seizures (status epilepticus; defined as a seizure lasting >30 min) by chemoconvulsants or electrical stimulation triggers rapid induction of inflammatory mediators in brain regions of seizure activity onset and propagation (Vezzani et al., 2000a, 2011; Crespel et al., 2002). Immunohistochemical studies on rodent brains after induction of status epilepticus demonstrated subsequent waves of inflammation during the epileptogenic process (that is, the process underlying the onset and chronic recurrence of spontaneous seizures after an initial precipitating event), involving various cell populations. Findings from these and other studies show that proinflammatory cytokines (IL-1β, TNF and IL-6) are first expressed in activated microglia and astrocytes, and cytokine receptor expression is up-regulated in microglia, astrocytes and neurons (Vezzani and Granata, 2005). These initial events are followed by the induction of cyclooxygenase-2 (COX-2) and, hence, prostaglandins, and up-regulation of components of the complement system in microglia, astrocytes and neurons (Yoshikawa et al., 2006; Aronica et al., 2007; Kulkarni and Dhir, 2009; Xu et al., 2009).

In addition to the molecules mentioned above, chemokines and their receptors are produced – predominantly in neurons and in activated astrocytes – days to weeks after status epilepticus (Wu et al., 2008; Xu et al., 2009; Fabene et al., 2010). An ensuing wave of inflammation is induced in brain endothelial cells by seizures, and includes up-regulation of IL-1β and its receptor IL-1R1, the complement system, and adhesion molecules (P-selectin, E-selectin, intercellular adhesion molecule 1 (ICAM) and vascular cell adhesion molecule 1) (Vezzani and Granata, 2005; Aronica et al., 2007; Fabene et al., 2008; Vezzani et al., 2011). The presumed cascade of events leading to this vascular inflammation involves seizure-induced activation of perivascular glia, which produce and release cytokines and prostaglandins. Importantly, no peripheral immune cells or blood-derived inflammatory molecules are required for vascular inflammation, as such events have been replicated in vitro in isolated guinea pig brain undergoing seizure activity (Vezzani and Granata, 2005; Vezzani et al., 2011).

The presence of inflammation originating from the brain might promote the recruitment of peripheral inflammatory cells. Indeed, chemokines expressed by neurons and glia and in the cerebrovasculature following seizures might direct blood leukocytes into the brain, which would be consistent with the reported emergence of granulocytes during epileptogenesis, and sparse T lymphocytes in chronic epileptic tissue from TLE models and humans (Ravizza et al., 2008). As in human epileptic brain specimens, brain tissue from rodents with experimental chronic TLE contains both activated astrocytes and microglia expressing inflammatory mediators (Crespel et al., 2002; Dube et al., 2007; Ravizza et al., 2008). Evidence for brain vessel inflammation associated with BBB breakdown is also prevalent (Fabene et al., 2008). A recent veterinary study evaluated the relationship of microglial activation to seizure-induced neuronal death in the cerebral cortex of Shetland sheepdogs with familial epilepsy (Sakurai et al., 2013). Cadavers of ten Shetland sheepdogs from the same family (six dogs with seizures and four dogs without seizures) and four age-matched unrelated Shetland sheepdogs were evaluated. Samples of brain tissues were collected after euthanasia and sectioned for H&E staining and immunohistochemical analysis. Evidence of seizure-induced neuronal death was detected exclusively in samples of cerebral cortical tissue from the dogs with familial epilepsy in which seizures had been observed. The seizure-induced neuronal death was restricted to tissues from the cingulate cortex and sulci surrounding the cerebral cortex. In almost the same locations as where seizure-induced neuronal death was identified, microvessels appeared longer and more tortuous and the number of microvessels was greater than in the dogs without seizures and control dogs. Immunohistochemical results for neurons and glial cells (astrocytes and microglia) were positive for vascular endothelial growth factor, and microglia positive for ionized calcium-binding adapter molecule 1 were activated (i.e. had swollen cell bodies and long processes) in almost all the same locations as where seizure-induced neuronal death was detected. Double-label immunofluorescence techniques revealed that the activated microglia had positive results for TNF-α, IL-6 and vascular endothelial growth factor receptor 1. These findings were not observed in the cerebrum of dogs without seizures, whether the dogs were from the same family as those with epilepsy or were unrelated to them. The suggested conclusion of this study was that microglial activation induced by vascular endothelial growth factor and associated pro-inflammatory cytokine production may accelerate seizure-induced neuronal death in dogs with epilepsy (Sakurai et al., 2013).

The findings discussed above show that brain inflammation induced by status epilepticus develops further during epileptogenesis and demonstrate that this phenomenon persists in chronic epileptic tissue, thereby supporting the idea that inflammation might be intrinsic to, and perhaps a biomarker of, the epileptogenic process (Dube et al., 2007).

Does inflammation cause seizures?

Although the functions of many inflammatory mediators remain unresolved, clear evidence exists for an active role for IL-1β, TNF, IL-6, prostaglandin E2 (PGE2) and the complement cascade in seizure generation and exacerbation (Xiong et al., 2003). Seizure activity leads to the production of inflammatory molecules that, in turn, affect seizure severity and recurrence, and this action takes place through mechanisms distinct from the transcriptional events traditionally activated during systemic inflammation. Cerebrospinal fluid studies in children and animal models have implicated the release of endogenous cytokines, especially IL-1β, in the generation of febrile seizures and, possibly, in the development of epilepsy after febrile seizures (Haspolat et al., 2002; Virta et al., 2002; Dube et al., 2005; Heida and Pittman, 2005; Vezzani et al., 2013).

A positive feedback pathway has been identified in rat models between seizure activity and the presence of inflammation (Vezzani et al., 2011). However, the role of inflammation in epilepsy in veterinary medicine has really only been described clinically in cats with hippocampal necrosis (Fatzer et al., 2000). Hippocampal lesions of 38 cats with seizures have been described and seemed to reflect different stages of disease consisting of acute neuronal degeneration to complete malacia, affecting mainly the layer of the large pyramidal cells but sometimes also the neurons of the dentate gyrus and the piriform lobe. The clinical, neuropathologic and epidemiologic findings suggest that the seizures in these cats were triggered by primary structural brain damage, perhaps resulting from excitotoxicity, but secondary inflammation cannot be ruled out in these cases.

Does inflammation cause cell loss?

Available studies suggest that seizure-related or injury-related inflammation might contribute to cell loss and synaptic reorganization, which are important mediators of the development of hyperexcitable circuits that lead to epilepsy after insults such as status epilepticus or TBI in the adult rodent brain (Bartfai and Schultzberg, 1993; Buckmaster and Dudek, 1997; Pitkanen and Sutula, 2002). Inflammation is induced rapidly following such insults, preceding neurodegeneration in lesional models of seizures (Rizzi et al., 2003; Ravizza and Vezzani, 2006). This finding is consistent with the idea that inflammation augments cell death, which is further supported by data from studies involving injection of inflammatory mediators together with excitotoxic stimuli (Allan et al., 2005). Activation of microglia and astrocytes and production of cytokines and PGE2 can occur in seizure models where cell loss is not detected in immature or adult rodents (Vezzani et al., 1999, 2000a; Rizzi et al., 2003; Kovacs et al., 2006; Dube et al., 2010). Such observations suggest that rather than being a consequence of cell loss, seizure-induced brain inflammation can contribute to cell death (Vezzani and Baram, 2007). Additional interactions between inflammation and cell death in the context of epilepsy have been observed. Brain injury, such as TBI, causes tissue inflammation that seems to contribute to both cell death and long-term hyperexcitability (Clausen et al., 2009; Longhi et al., 2009). In the context of CNS injury (for example, in chronic neurodegenerative diseases or acute stroke), inflammation can have a neuroprotective role (Liesz et al., 2009; Schwartz and Shechter, 2010). Indeed, whether micro-glia, macrophages and/or T cells are destructive or neuroprotective seems to depend on their activation status, which is orchestrated by the specific inflammatory environment (Rothwell, 1989; Schwartz and Shechter, 2010). This balance, together with the specific brain regions in which inflammation develops, might account for the relatively low incidence of seizures in other neurological disorders associated with brain inflammation (Vezzani et al., 2013).

Mechanistic insights

Several established and novel mechanisms could mediate the effects of inflammatory mediators on neuronal excitability and epilepsy. Some of these mechanisms could be involved in the precipitation and recurrence of seizures, while others are implicated in the development of epileptogenesis (Vezzani and Baram, 2007). These mechanisms constitute potential molecular targets for drug design, and are briefly summarized here. As discussed above, IL-1β and HMGB1 activate convergent signalling cascade through binding to IL-1R1 and TLR4, respectively (Akira et al., 2001; Perkins, 2007; Hoebe and Beutler, 2008). The downstream pathways activated by these ligands converge with the TNF pathways at the transcription factor NFκB, which regulates the synthesis of chemokines, cytokines, enzymes (for example, COX-2) and receptors (for example, TLRs, IL-1R1, and TNF p55 and p75 receptors) (Gilmore, 2006). This transcriptional pathway modulates the expression of genes involved in neurogenesis, cell death and survival, and in synaptic molecular reorganization and plasticity (processes that occur concomitantly with epileptogenesis in experimental models) (Buckmaster and Dudek, 1997; Pitkanen and Lukasiuk, 2009).

Immune and anti-inflammatory therapies

If immune mechanisms and inflammation do indeed have a role in the generation of seizures, immune-modulating and anti-inflammatory therapies might be effective treatments for some or all forms of epilepsy. Therapies such as ACTH, corticosteroids, plasmapheresis and intravenous immunoglobulin (IVIg) have been employed to treat seizures and/or epilepsy, with varying success. These therapies have all been employed in human patients with presumed autoimmune limbic encephalitis, where early and aggressive treatment often seems to be useful (Vincent et al., 2010).

The presumed mechanism of action of the therapeutic agents listed above is suppression of inflammation; however, other modes of action might also be involved, including direct effects on brain excitability, and suppression of endogenous proconvulsant brain agents (Baram and Hatalski, 1998; Joels and Baram, 2009).

The use of steroids in various forms is common for more severe, treatment-resistant forms of childhood epilepsy. ACTH, steroids and IVIg have all been employed to treat AEM-unresponsive paediatric epilepsies, difficult focal (partial) epilepsies and myoclonic epilepsies (You et al., 2008). Unfortunately, determination of whether patients received benefit from these treatments is problematic, since most of these epilepsies are extremely heterogeneous in aetiology and severity, and exhibit notoriously variable courses. In addition, most of the clinical studies are retrospective case series, with occasional prospective case series that lack controls (Mikati et al., 2002; Verhelst et al., 2005).

Follow-up duration in these case series was also often variable. A recent review of investigations of IVIg in intractable childhood epilepsy found no randomized or controlled studies and, in fact, only two case series employed statistics in assessing outcome (Mikati et al., 2010). One series showed a statistically significant reduction in seizures with IVIg treatments, while the other revealed an insignificant trend with such therapy (Mikati et al., 2010). However, a Cochrane Collaboration review on the use of ACTH for other childhood epilepsies, published in 2007, found only a single randomized controlled trial, which only included five patients (Gayatri et al., 2007). The authors of this review concluded that, at present, no evidence exists to support either the safety or the efficacy of ACTH for general paediatric epilepsies (Gayatri et al., 2007).

Disorders of Neuronal Migration and Seizures

The major developmental disorders noted in humans giving rise to epilepsy are disorders of neuronal migration that may have genetic or intrauterine causes (Engelborghs et al., 2000). Abnormal patterns of neuronal migration lead to various forms of agyria or pachygyria whereas lesser degrees of failure of neuronal migration induce neuronal heterotopia in the subcortical white matter. Experimental data suggest that cortical malformations can both form epileptogenic foci and alter brain development such that diffuse hyperexcitability of the cortical network occurs (Chevassusau-Louis et al., 1999). Other studies revealed increases in postsynaptic glutamate receptors and decreases in GABAA receptors in micro-gyric cortex, which could promote epileptogenesis (Jacobs et al., 1999).

Periventricular heterotopia is a human X-linked dominant disorder of cerebral cortical development. Mutations in the filamin 1 gene prevent migration of cerebral cortical neurons causing periventricular heterotopia (Fox et al., 1998). Affected females present with epilepsy whereas affected males die embryonically.

Lissencephaly is a brain malformation characterized by a paucity of gyral formation and a thickening of the cerebral cortex. It is presumed to occur secondary to incomplete migration of immature neurons to the cortical plate during fetal development (Saito et al., 2002). Lissencephaly is considered to be the most severe type of neuronal migration disorder compatible with survival. In humans, it is presumed to result from an arrest of neuronal migration at approximately 3 to 4 months (Dobyns et al., 1993). Once they exit the cell cycle in the periventricular proliferative zone, immature neurons must migrate to the cortical plate along radial glial fibres (Rakic, 1988). The six layers of the cerebral cortex are formed in an ‘inside out’ pattern, with early migrating neurons forming the deep layer and later migrating neurons passing their migratory predecessors to form the superficial layers. Interruption at any stage of the process of neuronal migration may result in the arrest of neurons in an intermediate position between the periventricular zone and the cortex (Saito et al., 2002). Such an interruption may be due to a genetic lack of appropriate molecular cues, or secondary to non-genetic influences such as in utero infection or ischaemia. Secondary influences are a more common mechanism for the related cortical malformation, polymicrogyria.

In humans, mutations of two genes, LIS1 (located on 17p13.3) and DCX (located on Xq22.3), have been found to account for the majority of cases (Pilz et al., 1998). Both of these genes have been shown to have roles in neuronal migration by their interactions with the neuron microtubule network (Gleeson et al., 1999a; Sapir et al., 1999). X-linked lissencephaly and double cortex syndrome is a disorder of neuronal migration documented in humans. Double cortex or subcortical band heterotopias often occur in females whereas more severe lissencephaly is found in affected males. A causal mutation in a gene called doublecortin has been identified (Gleeson et al., 1998). It was suggested that doublecortin acts as an intracellular signalling molecule critical for the migration of developing neurons (Allen and Walsh, 1999; Gleeson et al., 1999b). Lissencephaly has been documented in Lhasa apsos with histopathology indicating the condition to be very similar to that seen in people (Greene et al., 1976; Saito et al., 2002). This condition has also been documented in a mixed breed dog and together with either cerebellar hypoplasia in two wire-haired fox terriers and three Irish setters, with cyclopia in one German shepherd-mixed breed dog, or with microencephaly in the Korat breed of cat (Saito et al., 2002; Lee et al., 2011).

Although these disorders are relatively rare, studying the underlying pathophysio-logical mechanisms may shed light on the pathophysiology of more common epileptic syndromes.

How Do Seizures Stop?

Most seizures are self-limited, lasting no more than a few minutes. The persistence of a seizure lasting longer than several minutes is usually a cause for alarm as physiological mechanisms terminating the seizure may have failed. Why seizures typically do not continue indefinitely, and how intrinsic anti-convulsant mechanisms in the brain lead to seizure termination, are questions that potentially offer new avenues for developing novel treatments for epilepsy, as well as offering insights into brain autoregulatory mechanisms.

Mechanisms acting at the level of single neurons

Within a single neuron, prolonged depolarizations with sustained action-potential firing may be triggered by a brief depolarizing pulse, as in the paroxysmal depolarizing shift, or may be the result of sustained excitatory synaptic input from neighbouring neurons engaged in seizure activity (Ayala, 1983). Intrinsic mechanisms of seizure termination active in a single neuron, discussed below, include: the potassium currents activated by calcium and sodium entry; the loss of ionic gradients, particularly of potassium, leading first to depolarization with increased firing, followed by depolarization blockade of membrane firing and cessation of firing; and possibly the depletion of energy substrates locally, with the decline in adenosine triphosphate (ATP), resulting in cessation of neuronal firing.

Intracellular ion-activated potassium currents

The membrane after hyperpolarization that follows bursts of action potential discharge is the result, at least in part, of potassium currents activated by the entry of calcium and sodium. Increased calcium entry during the paroxysmal depolarizing shift, or as a result of the action of glutamate at the postsynaptic membrane, activates a calcium-dependent membrane potassium conductance that allows potassium efflux, membrane hyperpolarization and cessation of firing (Alger and Nicoll, 1980; Timofeev et al., 2004). Like calcium, sodium entry may also activate a sodium-dependent potassium current that reduces neuronal excitability by hyperpolarizing the membrane and increasing shunt conductance (Schwindt et al., 1989).

Transmembrane ion gradients

The effect of extracellular potassium is multi-faceted. Sustained potassium efflux increases extracellular potassium concentration, depolarizing the membrane and moving the intra-cellular voltage toward the threshold for sodium action potential firing. As extracellular potassium continues to accumulate, there is membrane depolarization and action potential firing increases. With further accumulation, the membrane potential becomes more depolarized than the firing threshold for sodium-action potentials, sodium channels inactivate, and neuronal firing ceases. In vitro experiments by Bikson et al. (2003) illustrate these effects of extracellular potassium accumulation. Electrographic seizure-like activity triggered in hippocampal slices by exposure to low-calcium artificial cerebro-spinal fluid (aCSF) manifested as recurrent periods of population firing followed by periods of electrographic silence lasting 12–18 s. The termination of each electrographic discharge by a period of electrographic silence resulted from transient increases in extracellular potassium to plateaus of approximately 12 mM. The depolarized state was maintained by the elevation of extracellular potassium and by the presence of persistent sodium channels that did not inactivate. Depolarization blockade-terminating seizure-like discharges have also been observed in neocortical slices in which GABA-ergic inhibition is partially blocked by picrotoxin (Pinto et al., 2005). Focal or localized increases in potassium may also trigger additional potassium release beyond the initial region of potassium accumulation. Shifts in extracellular potential, and oscillations seen at the end of hippocampal after-discharges, have been attributed to a rapid rise in extracellular potassium that triggers waves of astrocyte depolarization and a propagating rise in potassium that terminates neuronal firing (Bragin et al., 1997). In addition to its direct depolarizing effects, increased extracellular potassium may also indirectly result in membrane depolarization through the action of the potassium–chloride co-transporter KCC2. The rise in extracellular potassium can increase intracellular chloride, shifting the chloride reversal potential toward membrane depolarization. In the setting of increased intracellular chloride, the action of GABA to open chloride channels could enhance membrane depolarization to the point of becoming refractory to further firing of action potentials (Jin et al., 2005; Galanopoulou, 2007).

Extracellular calcium levels also change markedly during paroxysmal neuronal firing and may affect the efficiency of neuron-to-neuron spread of activity. Focal seizure activity results in a decline in extracellular calcium activity of approximately 50% (Heinemann et al., 1977). This decline may inhibit synaptic transmission because synaptic vesicle fusion and neurotransmitter release are dependent on entry of extracellular calcium (King et al., 2001; Cohen and Fields, 2004). Decline in extracellular calcium also potentially affects gap junction function as hemichannel opening increases in low calcium (Thimm et al., 2005).

Energy failure

Sustained neuronal activation also markedly increases energy, namely ATP, utilization to restore ion gradients across the membrane. In some neurons, the presence of an ATP-gated potassium channel (KATP) reduces neuronal activity when ATP levels decline intracellularly (Yamada et al., 2001). When the ATP level falls because energy utilization outpaces energy production, potassium channels open and produce membrane hyperpolarization. Indeed, knockout mice lacking functioning KATP channels experience a