Seizure Mechanisms and Vulnerability in the Developing Brain

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Chapter 39

John W. Swann

Tallie Z. Baram

Frances E. Jensen

Solomon L. Moshé

Introduction

Throughout life, continuing maturational and functional changes within the brain impact seizure susceptibility and epileptogenesis. Early life is characterized by periods of excessive excitability to metabolic, electrical, and chemical stimuli as well as an inability to limit or suppress the effects of such stimuli. In humans, the incidence of seizures is highest in the first months of life, and several types of seizure disorders occur exclusively in infancy or childhood. Age-specific differences are noted in terms of motor behaviors and electroencephalographic (EEG) patterns, sensitivity to anticonvulsant treatment, and outcome. For example, in young children—and particularly in infants and preterm neonates—behavioral and EEG phenotypes can be very subtle. Often, these seizures are missed, because of lack of motor phenomena, or considered generalized tonic–clonic seizures because the features indicating focality are not overtly present. Another feature typical of early partial seizures is variability in clinical manifestations between individual seizures; the seizure phenotype is very much a function of brain developmental state. Unlike in the adult brain, focal dysfunction in the young brain can produce multifocal seizures or result in seizures with bilateral manifestations that can be loosely described as “generalized” (e.g., infantile spasms).¹

Several factors, including altered permeability of the blood–brain barrier, continuing development of neurons and glia, and changes in neuronal connectivity contribute in many ways to developmental windows of increased seizure susceptibility. Such windows have been observed in all species in which studies of spontaneous or experimentally induced seizures have been performed, and these studies reveal differences that depend on developmental stage. To understand the mechanisms responsible for these age-related differences, it is necessary to examine developmental differences in experimental animal models of seizures. Comparisons to information from human studies can be invaluable and serve to validate the clinical significance of findings from animal studies. Animal models can also be useful in understanding the effects seizures have on brain development.

This chapter reviews experimental evidence from both in vivo and in vitro studies that demonstrate a critical period of heightened seizure susceptibility in early life. We also describe (a) age-dependent changes in the propensity for focal seizures with secondary generalization and in the expression of primary generalized seizures; (b) the age-dependent functionality of neuronal networks involved in the control of seizures, as demonstrated in animal models and human studies; and (c) the impact of seizures on brain development, including the circumstances under which early-life seizures may produce epilepsy and/or learning deficits in later life.

Critical Developmental Periods of Enhanced Seizure Susceptibility In Vivo and In Vitro

Experimental evidence shows that developmentally discrete periods of increased seizure susceptibility and expression exist. Several reviews of this topic are available.¹⁵⁴^,¹⁸³ The first period in the rat is during the first postnatal week, analogous to the preterm human infant; at this age, the immature rat brain exhibits an EEG pattern that can resemble the pattern of the preterm human.⁹¹ Michelson and colleagues¹¹⁶^,¹¹⁷ have found that urethane-anesthetized or freely moving 7-day-old rats have the highest threshold for hippocampal afterdischarges. Mares also found elevated thresholds for hippocampal ADs in 7-day-old rats.¹¹¹ Seven-day-old rats have long refractory periods following electrical hippocampal stimulations, compared with rats in the second or third postnatal week.¹¹ In vitro intracellular recordings from hippocampal and neocortical slices taken during the first postnatal week have shown that action potentials routinely have slower rising and falling phases.⁹⁸^,¹⁶⁶^,¹⁶⁷ When seizures are elicited, they are far less synchronized than those recorded from tissue taken from rats 2 to 3 weeks of age.⁹⁸^,¹⁶⁷^,¹⁸⁶ Similar data have been obtained in kittens, in which electrical stimulations elicited broad action potentials and repetitive discharging was infrequent.¹⁴²^,¹⁴³

During the second and third postnatal week of the rat—ages roughly corresponding to human infants and young children—peak susceptibility to focal seizures occurs. Indeed, the baseline EEG at these ages transitions from the patterns similar to the human neonate, to the infant and early childhood periods.⁹¹ This increased excitability has been demonstrated in whole-animal experiments including neocortical focal epileptogenesis,¹¹¹ amygdala kindling,¹²⁸ hippocampal kindling,⁷³^,¹¹⁸ hippocampal electrical stimulations,¹⁹³ hypoxia, hyperthermia, and systemic administration of chemoconvulsants.¹²⁷ In addition, at these same ages, numerous laboratories have demonstrated increased susceptibility to the induction of seizure-like activity using in vitro slice preparations. In most models, robust ictal events occur only during this critical period of seizures susceptibility. These ictal discharges can be readily induced by γ-aminobutyric acid (GABA)_A receptor antagonists, 4-aminopyridine, elevated extracellular potassium, hypoxia, and electrical stimulation.⁴³^,⁷⁷^,⁷⁸^,⁹¹^,¹⁸⁶ Thus, the increased susceptibility to seizures during the second and third weeks of life is not restricted to a single structure or to a specific model, and it probably represents a widespread phenomenon intrinsic to a variety (but likely not all) of neuronal networks in the immature brain.

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Mechanisms of Enhanced Seizure Susceptibility

Alterations in Synaptic Transmission

Glutamate.

The period of enhanced seizure susceptibility corresponds in time with a period of rapid axonal and dendritic outgrowth. The first 2 weeks of life in the rat and the first year of life in the human and primates are periods of dramatic increases in synaptic and spine density.⁷⁹^,⁸⁸^,¹⁴⁵ As the brain matures, an increase occurs in the number of excitatory synapses that use glutamate as their neurotransmitter.

Glutamate receptor subunit expression is developmentally regulated in a number of ways. There is prominent expression of the NR2B, NR2D, and NR3A subunits in rodents during the neonatal period, which gradually decreases over time, while simultaneously NR2A expression increases to as much as tenfold over levels of expression at birth.¹²⁰^,¹⁵⁸^,²¹³ The functional consequences of this “subunit switching” is that, prior to the increases in NR2A expression, a net increase in excitability may be possible: NR2B results in longer current decay times,⁶¹ whereas NR2D and NR3A expression are associated with minimal Mg sensitivity, thus resulting in an increase in both N-methyl-D-aspartate receptor (NMDAR) channel opening frequency and time.

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) expression is increasing simultaneously in the rat forebrain.¹⁵⁴^,¹⁵⁵ Furthermore, in early life, AMPAR expression is characterized by a relative lack of the GluR2 subunit in hippocampal and neocortical pyramidal neurons.¹⁰¹^,¹⁵⁴^,¹⁵⁵^,¹⁸⁷^,¹⁸⁸ The lack of GluR2 allows for AMPARs to be calcium permeable, thus increasing intracellular signaling associated with glutamate receptor activation. Thus, differences in subunit composition could contribute to excitability during the critical period of neuronal hyperexcitability.

A limited number of human tissue studies confirm similar patterns of glutamate subunit expression across development. The mRNA for the NMDA receptor NR2B is expressed at high levels on principal neurons in cortex and hippocampus in the first year of life, whereas NR2A expression first appears around 1 to 2 years of age.¹³⁰ AMPAR expression is also highly developmentally regulated in the human. Immunocytochemical and Western blot analyses show that the GluR2 subunit does not appear on principal neurons in cortical gray matter until late in the first year of life,¹⁷¹ suggesting that, like the rodent, immature human principal neurons express calcium-permeable AMPARs.

The changes in expression of molecular markers for glutamatergic synapses observed in early life in neocortex and hippocampus, as well as the number of synapses, most likely reflect an increase in both the number of excitatory fibers projecting from distant sites and the proliferation of local circuit connections. An example of the latter is excitatory axons that arise for CA₃ hippocampal pyramidal cells that make synaptic contacts with neighboring CA₃ neurons (both pyramidal cells and interneurons) and also project as Schaffer collaterals to CA₁ pyramidal cells. Studies on the maturation patterns of recurrent excitatory collaterals in CA₃ pyramidal cells⁷¹ show that axon arbors are very short during the first postnatal week and, on average, branch infrequently. However, by the second postnatal week an exuberant outgrowth of these axons occurs, and branch number increases dramatically. Following this outgrowth, axon arbors appear to remodel. By adulthood, half the branches are lost, but the remaining axons increase in length concomitant with the overall growth of the hippocampus. The number of presumed presynaptic terminals increases dramatically from week 1 to week 2 and then remains unchanged into adulthood.

Alterations in Synaptic Transmission

GABA

Age-dependent differences in GABA-mediated synaptic transmission could also contribute to enhanced seizure susceptibility. In the rodent, GABA_A receptors and glutamic acid decarboxylase (GAD) levels steadily increase until the third or fourth postnatal weeks, suggesting a relative lack of inhibitory tone in the immature brain compared with the adult.²⁷^,¹⁸⁵ Furthermore, a number of studies have shown that GABA is an excitatory neurotransmitter during early postnatal life.⁴² This time period, postnatal (P)0 to P5, as originally reported, precedes the period of enhanced seizure susceptibility. Paradoxical depolarizing actions of GABA have been shown to be due to differential chloride (Cl^-) homeostasis in immature neurons compared with adult.¹⁷^,¹⁴⁹ The immature neurons have high Cl^- concentrations since they lack the Cl^- extruding cotransporter KCC2, but have high expression of the Cl^- importer NKCC1.⁵⁷^,¹⁴⁹ Hence, GABA channel opening results in Cl^- outflow down the concentration gradient in the immature neuron, and hence to depolarization and neuronal excitation rather than the hyperpolarization and inhibition associated with Cl^- influx in mature cells. Rodent studies reveal that NKCC1 actually peaks during the first week of life, whereas KCC2 protein expression peaks between P5 and P10 (depending on rat strain) and does not reach adult levels until the end of the second week of life or even later.¹⁷^,¹⁴⁹ The dramatic onset of expression of the Cl^- transporter, KCC2, is thought to herald a shift in the Cl^- reversal potential to a more hyperpolarized state, below the resting membrane potential.¹⁵⁰ Thus, during a developmental window, when recurrent excitation has become quite robust, excitatory GABA-mediated synaptic transmission in some pyramidal cells may further enhance excitability.¹⁷^,⁶²^,¹⁴⁹ The relevance of Cl^- transporter maturation to the excitability of the immature brain is also supported by the fact that the NKCC1 inhibitor, bumetanide, can markedly attenuate seizures when administered to P7 rats.⁵⁷

Limited parallel human data is available regarding the development of GABA systems. In human neocortex, GABA_A receptors and GAD levels continue to increase into mid-childhood.¹³⁰ NKCC1 expression peaks around term.⁵⁷ In fact, KCC2 protein expression is not present in human neocortex until after 1 year of age, and rises to adult levels in early childhood.⁵⁷ Taken together, these observations suggest that, as in neonatal rodents, GABA inhibition is likely to be significantly reduced in human infancy and early childhood.

Alterations in Ion Channel Expression and Function

Developmental changes in the intrinsic properties of neurons and/or their responses to network input have been discovered and are governed to a large extent by the age-dependent expression patterns of ion channels. Indeed, work in both animal models and humans has highlighted the contribution of genetic and acquired changes in ion channel structure and function to states of hyperexcitability, seizure susceptibility, and frank epilepsy.¹³⁴^,¹⁵⁹^,¹⁸²^,²¹⁰

Sodium channels.

Of the 13 expressed mammalian sodium (Na⁺) channel genes, mutations in three subunits (Nav1.1, Nav1.2, β1) have been associated with epileptic phenotypes. The functional consequences of the nearly 200 different mutations identified in these genes are diverse,¹¹⁵ but remarkably, the temporal evolution of the associated seizures indicates that the developing central nervous system (CNS) is particularly intolerant to these variations of Na⁺ channel function. Seizures resulting from Na⁺channel dysfunction commence during infancy or early childhood, and often disappear later.¹⁶³ The reasons for the developmental susceptibility to Na⁺channel dysfunction are not clear. Although age-dependent expression patterns of Na⁺channel subunits have been described,¹⁴^,³¹^,⁷² they do not define vulnerability windows, when only a single isoform

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is expressed in the CNS, nor do they suggest maturation-related compensatory upregulation of nonaffected isoforms. Alternatively, the apparent age-specificity of these Na⁺-channel defects may be a result of age-specific insults that interact with these channels. For example, increased temperature (fever) may unmask specific deficits of mutated Na⁺ channels.¹⁷⁴ Consistent with this notion, sodium channel defects are particularly common in individuals with febrile seizures plus (FSP).¹⁶³

Potassium channel superfamily.

Developmental susceptibility to seizures that is governed by the function of potassium (K⁺) channels is evident from studies of dysfunction of two members, KCNQ2 and KCNQ3, that carry the M-current, a slowly activating outward current that regulates subthreshold excitability and prevents repetitive action potential firing.²¹⁸ Mutations in these lead to benign familial neonatal convulsions, seizures that occur during the first days and weeks of life and disappear later.⁴⁵ Whereas age-dependent expression of splice variants of these channels have been described,¹⁷¹^,¹⁹⁰ they seem to play little role in the temporal onset and resolution of the seizures. Intriguingly, the M-current, carried by these channels, may serve as a major inhibitory mechanism during the age when GABA is depolarizing,¹³⁶ so that even a 25% reduction of the current is sufficient to induce seizures,⁹³ explaining the occurrence of seizures neonatally. Once the role of GABA is reversed from depolarizing to inhibitory (e.g., the second postnatal week in the rat), a new powerful inhibitory system comes into play, so that persistent dysfunction of the KCNQ channels no longer renders the neuron hyperexci-table.

HCN (or h) channels, members of the K⁺ channel superfamily, contribute to the maintenance of resting membrane potential, integration of dendritic excitability, and neuronal depolarization in response to network input.¹³⁸^,¹⁵¹ The properties of the h channels are governed by the types of HCN channel isoforms that the neuron expresses, and the expression of HCN channel isoforms (1, 2, and 4) varies drastically as a function of age.¹⁸^,²⁶ Importantly, hippocampal maturation is associated with marked increase in the expression of the HCN1 isoform²⁶ that tends to dampen dendritic excitability and minimize rebound depolarization in response to hyperpolarizing input¹⁴⁰ and could contribute to reduced seizure susceptibility in the juvenile and adult brain.¹⁵⁹

Calcium channels.

Calcium (Ca²⁺) channels are involved in age-specific seizure vulnerability at a somewhat later age than the neonatal-infant period discussed here. In the human, dysfunction of members of the (Ca²⁺) channel superfamily (Cav2.1, Cav3.2, β4) may be involved in absence seizures.⁴¹^,⁵⁸^,⁹⁵ An elegant explanation for the age-specific contribution of the Cav2.1 subunit to seizure susceptibility has been provided in experimental models by Noebels.¹³⁴ In neonatal mouse thalamic neurons, neurotransmitter release is dependent on both N- and P/Q-type channels.⁸⁹ With maturation, this function is taken over exclusively by the P/Q-type channels, formed by Cav2.1 subunits, so that dysfunction of these channels provoked seizure vulnerability.

The understanding of the contribution of intrinsic ion channels to age-dependent seizure vulnerability is rapidly evolving. New ion channels are being discovered that are expressed in an age-dependent manner and may enhance excitability in developing neurons (e.g., NKCC1),⁵⁷ thus further highlighting the importance of function and dysfunction of intrinsic ion channels to seizure susceptibility early in life.

Alterations in Peptide Neurotransmitters

Neuropeptides are released from neurons and can influence the excitability of a neuronal network through metabotropic receptors at post- or presynaptic sites (or both). Neuropeptide Y (NPY) is expressed in developing and adult rodent hippocampus in modest amounts.⁵^,⁸⁰^,¹⁶⁸ Interestingly, it is also found in human hippocampus, starting prenatally and persisting to adulthood.¹⁰⁸^,²¹⁷ NPY, acting via the Y2 (and possibly Y5) receptors, reduces network excitability and seizure susceptibility.⁴⁴^,²⁰⁵ Whether the relatively low levels of NPY during early development⁵ contribute to the enhanced vulnerability of the hippocampus is unknown.

Corticotropin releasing factor (CRF) or hormone (CRH) is an excitatory neuropeptide that reduces spike afterhyper-polarizations,⁴ and interacts with glutamatergic neurotransmission to promote excitability in vitro.¹⁰^,⁸³ In vivo, the administration of CRH causes age-specific seizures, especially when infused in immature hippocampus. Hippocampal CRH receptors peak during the first 2 postnatal weeks in hippocampus and amygdala.¹⁰ Interestingly, endogenous CRF is much more abundant in developing compared with mature hippocampus.³⁹ Thus, the actions of the endogenous peptide (which is released during stress)⁴⁰ favor increased excitability and seizure vulnerability during development.

Age-Dependent Alterations in Patterns of Seizure Propagation

Kindling is one of the best models of epilepsy to study the patterns of seizure propagation.⁷⁰ Kindling, once induced, permanently changes the susceptibility of the brain to seizures.⁷⁰ Amygdala or hippocampal kindling can be produced in 8- to 15-day-old pups using frequent stimulations (e.g., every 15 minutes).¹⁰⁵^,¹²³ In adults, stimulations delivered every 15 minutes either significantly retard or fail to induce kindling.⁷⁰^,¹²³^,¹³⁹^,¹⁴⁴ Progression through the various seizures stages in young animals is different from kindled seizure stages in prepubescent and adult rats. Stages 0 to 2 represent local events, stage 3 the involvement of the hemisphere ipsilateral to the stimulation site, stages 4 to 5 bilateral (generalized) seizures, while stages 6 and 7⁷⁴ may reflect spread to the brainstem.²⁹^,³²^,⁶⁴ Compared to older rats, pups spend proportionally less time in the early stages of kindling (stages 0–2) that are associated with focal seizures.¹²⁸ Instead, there is an early appearance of bilateral, although often asynchronous, seizures, indicating a tendency for seizure generalization. Pups experience many stage 3 to 4 seizures intermixed with isolated stage 5 seizures, followed by the explosive onset of stage 6 and stage 7 seizures.⁷⁴ Spontaneous seizures occur more readily in pups compared with adults.¹¹^,⁷⁵

Another difference between adults rats and 15-day-old rat pups involves the phenomenon of kindling antagonism.⁷ In adult rats, concurrent kindling of two limbic foci results in the suppression of generalized seizures from one or both sites. Pups do not show kindling antagonism to the development of generalized seizures between amygdala and hippocampus, or between the amygdala.⁷⁴ These data may indicate that, early in life, different brain areas can mutually enhance their epileptogenic potential and lead to the development of multifocal epilepsy, a common clinical phenomenon in young children, especially in those with infantile spasms. The data also suggest that, during the critical period, the immature CNS is more prone to the development of secondary generalized seizures. Thus, increases in seizure susceptibility extend beyond the local generation of epileptic discharges and involve mechanisms of seizure propagation to additional structures.

Kainic acid (KA) is commonly employed to induce focal seizures with secondary generalization throughout postnatal life. Administration of this excitatory amino acid leads to the development of seizures in all ages; however, the seizure manifestations are age-dependent, with specific manifestations such as scratching and swimming-like movements occurring in rats less than 15 days of age, while “wet dog shakes” are rare in

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these ages.³ Tonic–clonic seizures are regularly elicited in 7- to 25-day-old rats; however, their frequency decreases with age, especially after the third postnatal week. Rat pups are also more prone to develop status epilepticus (SE) than adult rats. In all age groups, EEG seizures start in the hippocampus; however, in young rats rapid involvement of the cortex occurs.

Age-Dependent Differences in Animal Models of Generalized Seizures

Most developmental models of generalized, predominantly motor seizures rely on the systemic administration of a chemoconvulsant, usually a GABA-related substance. The most commonly used agents are pentylenetetrazol (PTZ), bicuculline, or picrotoxin, and glutamate agonists such as kainite.¹²⁶^,¹⁹²^,¹⁹⁵ After day 7, the seizures consist of motionless state (freezing), myoclonic twitches, face and forelimb clonus, and generalized tonic–clonic movements. These seizure types, their latency to seizure onset, and their EEG correlates are dose dependent. The EEG correlate of motionless stare are rhythmic spike-and-wave discharges in a spindle-shaped envelope. Myoclonic body twitches are usually associated with isolated spike-and-wave discharges. Clonic and tonic–clonic seizures may have similar EEG correlates consisting of fast multiple spike-and-wave discharges. The chemoconvulsant-induced phenomena are thought to be the models of different human seizures. The motionless state and its associated rhythmic EEG activity is considered to be the model of human absences. Clonic seizures are considered a model of human myoclonic seizures, whereas tonic–clonic seizures may represent a model of primary generalized tonic-clonic seizures.

The expression of drug-induced motor seizures is age-dependent.¹⁹² Thus, with PTZ, clonic motor seizures are not often observed in rat pups prior the second postnatal week, rather swimming-like movements occur, reflecting an immaturity of the motor pathways; the CD₅₀ for clonic seizures remains constant after the third postnatal week. Myoclonic twitches and tonic–clonic seizures occur throughout development, but the CD₅₀ increases progressively with age. In rats less than 7 days old, the only seizure manifestations may be swimming movements of all four limbs, not accompanied by any EEG correlates. EEG discharges can be dissociated from behavioral seizures, and the duration of the epileptiform discharges decreases with age. Sharp waves become spikes after the second postnatal week, whereas the onset of electrographic seizures is synchronized in all areas after the third postnatal week.¹⁶⁵ With flurothyl, the seizure threshold increases with age. The clonic seizures become more apparent as the animal reaches the second week of life. During the first two postnatal weeks, clonic seizures in the majority of the models rapidly progress into tonic–clonic seizures. Thus, clonic seizures may be masked by tonic–clonic seizures.²⁰⁰^,²⁰³ One reason for the dissociation between behavioral and electrographic seizures may be the lack of cortical involvement in the expression of seizures in developing animals. Metabolic studies using the deoxyglucose technique have revealed that, in adults during clonic seizures, metabolic activity increases in the cortex, hippocampus, globus pallidus, and substantia nigra (SN). During tonic–clonic seizures, metabolic activity increases also in the midbrain structures.¹⁶ In contrast, in developing animals, decreases in neocortical activity occur with increases of activity in brainstem structures.¹²⁶^,¹⁷⁵^,¹⁹² One notable exception is the lack of activation of the SN, a structure thought to play an important role in the control of generalized seizure.¹²⁵

Generalized seizures can also be induced by electrical stimulation (electroshock seizures). Depending on the intensity the stimulating current, two types of seizures may occur:¹¹⁰ minimal clonic seizures involving clonic movements of the head and forelimbs and maximal, generalized, tonic–clonic seizures with a loss of righting reflexes. Minimal seizures probably represent a model of myoclonic seizures, and are generated in the forebrain.¹⁰⁷ Maximal electroshock seizures (MES) are a model of generalized tonic–clonic seizures, and involve brainstem structures.²⁸ The mature pattern of MES emerges during the third postnatal week in the rat; younger animals show only forelimb flexion (PN 10–12 or earlier), or forelimb flexion followed by forelimb extension and hindlimb flexion (PN 13–15).²⁰⁴

Drug-induced models of absence seizures also are available.⁴⁶ The acute pharmacologic models of typical absence seizures are induced from systemic administration of a single pharmacologic compound [4,5,6,7 tetrahydroxyisoxazolo (4,5,c) pyridine 3-ol (THIP), γ-butyrolactone, PTZ, or penicillin]. With appropriate doses, their administration leads to bilaterally synchronous spike-and-wave discharges associated with behavioral arrest, facial myoclonus, and twitching of the vibrissae. An acquired chronic model of atypical absence seizures has been derived from a timely prenatal administration of methylazoxymethanol (MAM) in combination with postnatal systemic administration of an inhibitor of cholesterol, AY-9944. For this seizure type, genetic models of absence epilepsy also are available, such as the GAERS and WAG/Rij rats, as well as various mouse mutants.¹³³

Age-Dependent Activity of Brain Networks Mediating Seizure Control

Circuits involving several subcortical nuclei can regulate seizures. One such system includes a GABA-sensitive SN-based circuit. The SN and especially the pars reticulata (SNR) may be critically involved in the expression and control of bilateral, generalized seizures in rats.¹²⁵ The nigral effects are operative in both seizures of local origin (kindling) and seizures that appear to be generalized from their onset (flurothyl-induced seizures). These nigral effects on seizures are age- and sex-specific¹⁹⁶ and appear to involve two distinct regions within the SNR, SNR_anterior and SNR_posterior. The segregation occurs with maturation; before the third week of life, only one functional SNR region is present. Both GABAergic and glutamatergic systems are involved, with ample information on GABA_A-mediated neurotransmission available.²²^,²³^,⁵¹^,⁶³^,⁶⁵^,⁶⁶^,⁶⁷^,¹¹³^,¹¹⁴^,¹²⁴^,¹²⁵^,¹⁹⁶^,¹⁹⁸^,²⁰² In male adult rats, bilateral microinfusions of muscimol into the SNR_anterior produce anticonvulsant effects, whereas microinfusions into the SNR_posterior are proconvulsant.¹²² In male immature (less than 25 days old) rats, there is no regional compartmentalization for the SNR effects on seizures, and muscimol infusions produce only proconvulsant effects. Studies in female rats show that the effects of the SNR on seizures are sex-specific with differences in the maturation patterns and a universal lack of SNR-mediated proconvulsant effects following muscimol microinfusions.¹⁹⁹ Maturational changes in electrophysiologic neuronal properties, in subunit composition of GABA_A receptors, expression of KCC2,⁶² and in the output targets may each contribute to the emergence of pro- and anticonvulsant regions within the SNR.¹⁹⁴^,¹⁹⁷^,¹⁹⁸^,²⁰² The sex-specific differences appear to be under the control of postnatal testosterone and its metabolites.^69a^,¹⁹⁹^,²⁰⁰

Impact of Early-Life Seizures: Chronic Hyperexcitability and Epilepsy

Early-Life Status Epilepticus

Animal models have contributed significant data toward the understanding of SE–related sequelae, and they offer the

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opportunity to explore cellular, molecular, and functional changes following status as function of age and sex. In models, the pre-existing substrate is known, and thus changes occurring following status can be considered causally related to the status per se, although the means by which status was induced may have an impact. Combining different data sets can identify common model-independent findings. The consequence can vary depending on whether status is induced in animals with normal or abnormal brains.

Studies in animals with normal brains have shown that the immature brain is relatively resistant to SE-induced morphologic damage.⁹^,³⁴^,¹⁸³^,²⁰¹ Long-lasting status, regardless of seizure origin (nonlimbic or limbic), induced by flurothyl,¹⁷⁶^,²⁰⁸ pentylenetetrazol,¹³¹ NMDA,¹⁷⁸ or KA,³^,¹⁵^,⁷⁶^,¹⁷⁹ and pilocarpine³⁴ is associated with little or no neuronal damage in the hippocampus or extrahippocampal regions in rats younger than 3 weeks. Synaptic reorganization in the supragranular layer of the dentate gyrus of the hippocampus does not occur following status before the third postnatal week.¹⁹^,⁷⁶^,¹⁵⁷^,¹⁷⁷. Many factors may contribute to the relative resistance of immature hippocampus to status-induced damage.⁸¹ With maturation, the extent of hippocampal damage increases.³^,⁸⁵^,¹⁵⁷

It should be noted that several groups suggest that a degree of hippocampal or extrahippocampal injury may occur after SE during early development.¹²^,⁴⁷^,⁴⁸^,⁷⁶^,⁹⁹^,¹⁴⁸^,¹⁵⁶^,¹⁵⁷^,¹⁷⁹^,¹⁸⁹^,²⁰⁹ However, the extent of damage is far less prominent compared to status-induced changes in adulthood. Status early in life may also prime the brain to the effects of subsequent insults.⁶⁹^,⁹⁷ These consequences may be either detrimental to the brain or, in some cases beneficial, as is the case with the effects of 1-hour-long flurothyl status on subsequent focal ischemic insult later in life.⁶⁹

The relationship of status to the subsequent development of epilepsy in normal immature brain is also age-dependent. In rats younger than P21 days, spontaneous seizures do not occur following KA, and are rarely observed following lithium/pilocarpine-induced SE.¹⁵⁷ In 21-day-old rats, spontaneous seizures occur irregularly in 10% to 73% of animals, depending on the seizure model used.¹⁴¹^,¹⁵²^,¹⁷⁹^,²⁰⁷ In adult rats exposed to status, the incidence of seizures reaches almost 100%.¹⁷⁹^,²⁰⁷ Furthermore, 15-day-old rats exposed to KA-induced status are not more prone to develop amygdala-kindled seizures as adults than are controls; this is not the case in adults.¹³⁷ MRI changes identified within 48 hours after status can predict which 21-day-old rats will develop seizures.¹⁵² Only rats with visible structural MRI abnormalities or without visible abnormalities, but with changes in T2 relation time, developed spontaneous seizures. Not all rats with epilepsy showed hippocampal damage. Three episodes of pilocarpine SE in 7- to 9-day-old rats produced long-term changes in epileptogenesis.¹⁶⁰

Very few studies assess whether SE-induced neuronal damage may be enhanced in the setting of a compromised brain in developing rats. These studies seem to suggest that, in this scenario of a two-hit hypothesis, an augmentation of hippocampal injury occurs following status.¹⁰⁴^,¹²²^,²¹⁵

With regard to two-hit models, a recent study by Scantlebury et al.¹⁶² reported that hyperthermic seizures induced in P10 rats with a single freeze-lesion resulted in 86% of rats with spontaneous limbic seizures. Controls with freeze lesions were not epileptic, and a minority of those with hyperthermic seizures had abnormal EEGs.

Febrile Seizures

Febrile seizures (FS) are the most common type of seizures in infants and young children,¹⁸¹ and it is important to understand their impact on the developing brain and potential contribution to epileptogenesis. To probe directly the mechanisms by which these seizures might contribute to temporal lobe epilepsy (TLE), several models are available, including one employing hyperthermia-induced seizures of 20-minute-duration evoked on postnatal day 10,⁵²^,⁵³^,⁵⁴^,⁵⁵^,⁵⁶ and those using heated water⁶⁸^,¹²¹ or heated copper sheets.⁸⁶

Early-life experimental FS produce a chronic state of hyperexcitability in an otherwise neurologically normal brain. This is manifest in vivo as an increased seizure susceptibility in animals experiencing experimental FS to convulsants given later in life, as compared with naive or hyperthermic controls,⁵² the presence of interictal discharges in hippocampal EEGs in 88% of adult rats that had experienced prolonged FS early in life,⁵³ and the emergence of spontaneous seizures in a proportion (∼35%) of FS-experiencing rats.⁵³ In vitro studies further localized this long-lasting hyperexcitability to the hippocampal circuit, despite increased inhibitory drive onto CA1 pyramidal cells.³⁷^,⁵²

These findings raised the question of the mechanisms by which prolonged FS febrile seizures may convert a “normal” developing brain into a hyperexcitable or proepileptic state. The underlying mechanisms are not yet fully understood, but available evidence indicates that subtle changes of network properties rather than gross morphologic alterations play a role. Experimental prolonged FS do not kill neurons.²⁰^,⁵³^,⁵⁵^,¹⁹¹ However, these seizures led to a significant and long-lasting change in the expression pattern of a specific ion channel, the hyperpolarization-activated cyclic adenosine monophosphate (cAMP)-regulated (HCN) channel (see the earlier discussion on ion channels). This “acquired channelopathy” consists of coordinated, enduring changes in the expression of several subunits of this channel family, altering the current they conduct, I_h, in hippocampal pyramidal cells.²⁴^,¹⁵¹^,¹⁵⁹ The mechanisms by which these changes promote hyperexcitability are discussed elsewhere.³⁶^,¹⁵⁹ It should be noted that, whereas transient changes in the pattern of HCN expression are found also af-ter kainate-evoked SE in the P10 rat, enduring transcriptional as well as posttranslational dysregulation of the HCN channels are unique to experimental FS,²⁵ and may derive from the involvement of cytokines in these, but not other seizures, during this stage of development.⁵⁴ Striking changes of the HCN channels are also found in human hippocampus from individuals with severe TLE and hippocampal sclerosis.¹⁹

Early-Life Hypoxia-Induced Seizures

Hypoxic encephalopathy is the major cause of neonatal seizures in the human infant.² Hypoxia-induced seizures can be associated with later-life neurocognitive effects and epilepsy.²¹^,⁶⁰^,¹³² Rats show a similar susceptibility to the epileptogenic effects of seizures in the immature period.¹⁵⁴

The exposure of rats to a brief period (15 min) of global hypoxia (5%–6% O₂) around P7 to P12, depending on strain, results in both behavioral and electrographic seizures.⁹¹^,⁹² In addition, these rats exhibit increased susceptibility to chemoconvulsant seizures later in life.⁹²^,⁹⁶ Hypoxic seizures are not associated with either immediate or subacute neuronal death in forebrain structures.⁹²^,⁹⁶ However, a number of alterations in neurotransmitter receptors and signaling pathways are observed in the surviving hippocampal and cortical principal neurons. Both the mRNA and protein for the GluR2 subunit appear to downregulate within 1 to 2 days following hypoxic seizures at P10, suggesting an increase in the number and activity of calcium-permeable AMPARs.¹⁵⁵ In addition, activation of existing calcium-permeable receptors by hypoxia in turn activates a number of signaling cascades that result in posttranslational receptor modification.¹⁵³ Within minutes following a seizure in a P10 rat, AMPAR activation of the phosphatase calcineurin by calcium causes dephosphorylation of GABA_A receptors and a decrease in inhibition.¹⁵³ Decreased inhibition due to GABA dephosphorylation and an increase in calcium

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permeability due to downregulation of GluR2 may contribute to the enhanced network excitability seen in the hours to days following hypoxic seizures. It is likely that these changes are not specific to hypoxia, but that alterations of neuronal function and molecular structure occur in surviving neurons in the immature brain, given the lack of neuronal death.

Brief but Recurring Seizures

In addition to SE and seizures induced by hypoxia/ischemic episodes at birth, a clinical condition often encountered in young children is brief but recurring seizures. This is particularly true in children who are unresponsive to anticonvulsant medication. Thus studies have been undertaken to determine the effects recurrent seizures have on brain development. Particularly important is the question whether recurrent seizures in early life contribute to the epileptogenic process.¹³ Do periods of repetitive seizures in early life increase neuronal excitability and/or produce epilepsy? To address this question, several animal models have been used. The tetanus toxin model, which was introduced a number of years ago in adult rats,⁹⁰ has been employed in immature animals.¹⁰³ Seizures are induced by a single unilateral stereotaxic injection of a very small quantity of tetanus toxin into the dorsal hippocampus on postnatal day 10. Tetanus toxin is known to block transmitter release by the proteolytic degradation of the synaptic vesicle docking protein, synaptobrevin.⁴⁹ However, tetanus toxin acts preferentially on inhibitory nerve terminals.²¹⁴ It is thought that by the selective blockade of GABA release, tetanus toxin is able to induce seizures. Tetanus toxin–induced seizures in infant rats begin 24 to 48 hours after hippocampal injections. Long-term video-EEG (V-EEG) recording has shown that rat pups experience brief (30–120 sec) but recurring seizures. Seizure frequency peaks 5 to 6 days after injections, then rapidly declines. Rats are likely to experience as many as 50 to 100 seizures in the week following tetanus toxin injection.¹⁴⁷

A number of studies of the tetanus toxin model have been carried out to characterize the effects of recurring early-life seizures. In this model, brief but recurring early-life seizures produce a chronic state of hyperexcitability that is most often characterized by interictal spiking on the EEG and in hippocampal slice recordings.¹⁰²^,¹⁰³^,¹⁷² Spontaneous, electrographic seizures are far less frequent, commonly do not have a behavioral component, and likely occur in rats that have experienced very frequent seizures in early life.⁶ Nonetheless, all animals appear to have an increased propensity for seizures when exposed to a convulsant.

The flurothyl model is a second model that has often been used to induce recurrent seizures in early life. Rats or mice are exposed to this volatile convulsant in an enclosed chamber. Flurothyl is thought to produce seizures by opening Na⁺ channels diffusely and possibly by blocking GABA_A receptors.²⁰⁶ In most studies, between 25 and 50 seizures have been induced in infant rats. Spontaneous behavioral seizures have not been reported later in life in flurothyl-treated animals. Epileptiform activity has not been observed on EEG recordings.⁵⁰ However, significant decreases in seizure susceptibility have been reported later in life when the threshold to flurothyl⁸⁷^,¹⁷³ or pentylenetetrazol⁸⁴ have been examined. Thus, as in the tetanus toxin model, flurothyl-induced seizures appear to increase neuronal excitability long-term. However, the degree of hyperexcitabilty does not appear to be sufficient to result in overt epilepsy or interictal spikes on the EEG.

Impact of Early-Life Seizures: Learning and Memory Deficits

The catastrophic epilepsies of infancy and early childhood are often associated with learning deficits, including mental retardation.³³^,⁸²^,¹³⁵^,¹⁷⁰ Infantile spasms and Lennox-Gastaut syndrome (LGS) are among numerous severe childhood seizure disorders characterized by frequent seizures. It is not uncommon for a child to be developmentally normal before the onset of such seizures, only to have his cognitive abilities fail to progress or even regress in the face of unremitting seizures. This has led many to wonder if seizures contribute to cognitive decline.¹¹² However, it remains controversial if early-life seizures hinder learning and the formation of memories, since other factors, such as an accompanying neuropathology or anticonvulsant therapy, could be responsible for cognitive problems. However, recent results from numerous animal studies support the notion that early-life seizures may impair learning, although it was much different from that of seizures in adults.

Status Epilepticus

Since the mid 1980s, a large number of studies have examined the long-term cognitive effects of SE in the developing rats.¹⁸⁰ These studies varied greatly in the way in which seizures were evoked, the age at which they were induced, the age at which behavioral testing was undertaken, and the behavioral tests employed to evaluate outcome. Thus, comparison of results can be difficult. Nonetheless, in general, results reported suggest that SE in early life does not have as severe impact on cognition—particularly spatial learning and memory—as similar seizures in older animals, starting from 21 days of age.¹⁰⁰^,¹⁰⁹^,¹⁶¹ One explanation for this age-dependency could be that SE incrementally induces neuronal cell death—particularly in hippocampus—after 21 days of age in the rat.¹⁰⁹ Neuronal cell loss, especially in the hippocampus, would be expected to lead to spatial learning and memory deficits. Since neuronal loss has been more rarely reported during the first 2 to 3 weeks (see previous discussion) of life, the acquisition of spatial memories might not be expected to be as severely impaired. Nevertheless, although a prolonged seizure in early life may not kill neurons in the CNS, they appear to produce other cellular and molecular changes that result in life-long deficits in learning and memory.¹⁰⁹^,¹⁶¹

Brief Recurring Seizures

Unlike studies of SE, the effects of brief but recurring early-life seizures have consistently shown that they produce spatial learning and memory deficits later in life.¹³ The majority of reports of memory and learning deficits following recurrent early-life seizures come from studies of the flurothyl model.⁵⁰^,⁸⁴^,⁸⁷^,¹⁰⁶^,¹⁷³ In most studies, seizures were induced beginning on the day of birth and extended through the first week of life; in some studies, seizure induction extended to postnatal 9 or 11, in order to increase the number of seizures evoked. In some studies, as few as 15 seizures were elicited; in others as many as 55. Rats could be tested for behavioral deficits as early as P24 or as late as P82. The Morris water maze and a subsequent probe test were used to assess spatial learning and memory. In all studies, the authors conclude that learning and memory were impaired. By comparing outcomes from several publications it would appear that the more seizures a rat experiences as a neonate, the more robust are the behavioral effects.

The behavioral effects of tetanus toxin–induced seizures in early life have been reported to be quite robust also and comparable to the most dramatic reports from the flurothyl model.¹⁰² This might be expected, because after a single injection of tetanus toxin, rats can experience as many as 50 to 100 seizures.¹⁴⁶ When these rats were 2 months of age, learning was compared across the three groups in the Morris water maze. The rats that had experienced recurrent seizures in early life were found to be markedly deficient in their ability to learn when compared with control groups. One possible confounding factor in studies of the tetanus toxin model is that,

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as adults, a significant fraction of the rats display epileptiform activity on the EEG (see earlier discussion). Subclinical seizures or even interictal spiking may interfere with the ability of neuronal networks to acquire new memories. To address this issue, EEG recordings were undertaken after rats were trained in a water maze.¹⁰² Results showed that rats without interictal spikes were just as impaired in their spatial learning abilities as those that had interictal spikes. Thus, the presence of interictal discharging could not explain the poor performance in this spatial learning task. The results are also in full accord with results from the flurothyl model, in which rats have been shown not to display epileptiform activity but are learning impaired.

A febrile seizure model in which rat pups were repeatedly made hyperthermic on P10 to P12 is a third model that has been employed recently to study the effects of brief but recurrent seizures.³⁵ Nine seizures were induced (three each day) that were 4 to 5 minutes in duration. The Morris water maze and probe test were used to access impaired cognition beginning on P60 and, as in all the other studies reviewed earlier, rats that had experienced recurrent seizures were cognitively impaired. Additional experiments showed that the learning deficits were not produced by hyperthermia but depended on the presence of recurring seizures. Thus, results from three different animal models are fully in accord with each other and suggest that brief but recurring seizures in early life can impair an animal’s ability to acquire new memories.

Underlying Mechanisms

Because neuronal death does not appear to contribute to the learning deficits in rats that have experienced seizures in infancy, identification of other potential mechanisms is important. One possible clue comes from a neuroanatomic study of hippocampal pyramidal cells in the tetanus toxin model of early-onset epilepsy.⁹⁴ Marked alterations were observed in the dendrites of CA3 neurons, which included a reduction in branching complexity of basilar dendrites as well as a decrease in spine density on both the apical and basilar dendrites. Similar observations have been made in human tissue obtained during epilepsy surgery.¹⁸⁴ In studies of hippocampal and neocortical foci, a decrease in length and branching complexity of dendritic arbors was observed, as well as a reduction in spine density on the remaining dendrites.¹²⁹^,¹⁶⁴ Similar observations have been made in the chronic alumina cream model of epilepsy in primates.²¹² Because dendrites and dendritic spines are sites of excitatory synaptic input onto neurons, the results suggest that glutamatergic synaptic transmission may be reduced. Moreover, because these synapses are recognized sites of activity-dependent alterations in synaptic transmission (e.g., long-term potentiation [LTP]), that are thought to underlie learning and memory,

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¹⁶⁹ studies that examine how seizures reduce dendritic arbor complexity in the brain could be an important step in understanding the mechanisms responsible for learning and memory deficits associated with early-onset epilepsy.

Two recent lines of investigations are beginning to demonstrate how seizures may impair dendritic structure and function. The first is based on the fact that seizures in early life are stressful and activate stress-mechanisms, including the secretion of the excitatory neuropeptide CRH from hippocampal neurons.⁴⁰ As mentioned earlier, both the peptide and its receptor are abundant in developing hippocampus.⁸^,³⁹ Recently, Chen et al. found that even in picomolar amounts, CRH interferes with dendritic growth and differentiation.³⁸ Mice lacking the CRH receptor had exuberant dendritic trees, whereas hippocampal pyramidal cells exposed to CRH during the first week of life had “atrophied” dendrites. The relevance of the stress-evoked, CRH-mediated damage to dendritic structure for cognitive deficits was highlighted in a recent report.³⁰ Early-life stress led to strikingly impoverished dendritic trees of hippocampal neurons later in life, and this was accompanied by learning and memory impairment, as well as reduced synaptic plasticity (LTP). The possibility that CRH may contribute to seizure-related dendritic atrophy is exciting, because antagonists for this peptide exist, and may prove useful to prevent or even reverse these deficits.⁵⁹

A second line of investigation emerges from the study of the effects of recurrent febrile seizures. In this study, investigators not only showed that seizures resulted in impaired learning and memory but also disrupted signaling that normally results in activation of the transcription factor, CREB.³⁵ When rats are tested in an inhibitory avoidance learning paradigm, normally an activation of CREB occurs by phosphorylation at Ser133. However, the investigators found that this activation is impaired after recurrent febrile seizures. This suggests a seizure-induced modification of a signally cascade upstream of CREB. To explore this possibility, the investigators treated adult rats that had experienced febrile seizures in infancy with rolipram, a specific phosphodiesterase type IV inhibitor, which results in the activation of protein kinase A (PKA) and is known to activate CREB via the mitogen-activated protien kinase (MAPK) pathway. Rolipram treatment was able to reverse the learning deficits observed in rats that had experienced recurrent febrile seizures. Because the MAPK pathway and CREB signaling are both thought to play key roles in dendritic development,¹¹⁹^,²¹⁶ it will be important to know from future studies where in the PKA-MAPK-CREB cascade that signaling is disrupted and if these changes are initiated very early in life by seizures and possibly contribute to the dendritic abnormalities observed.

Summary and Conclusions

Critical periods of enhanced seizure susceptibility have been identified in both man and animals, and mounting evidence suggests they share underlying mechanisms. It is unlikely that only one developmental process is responsible for the marked seizure susceptibility in early life. Instead, highly dynamic alterations in synapses and ion channels temporarily converge to produce neuronal network hyperexcitability. For example, during the second postnatal week in rodents, seizure susceptibility likely arises from a transient overlap between newly formed glutamatergic networks and the remaining excitatory GABAergic systems of the neonate that have yet to disappear. The interplay between these synaptic changes and coincident developmental alterations in ion channel function will be the subject for future studies.

In addition to similarities in seizure susceptibility, animal models of experimentally induced seizures share much in common with observations made clinically. While electrical and chemoconvulsive seizures may not be considered models of chronic epilepsy, they reproduce many of the age-specific electrographic and behavioral features of seizures observed in neonates, infants, and young children, and much has been learned from these models, especially in terms of understanding circuits underlying seizure propagation and control.

In recent years, numerous chronic models have been developed. They share features in common—like precipitation by an experimental induced seizure(s) in neonatal life or infancy in normal animals or, more recently, in animals with a brain malformation—the so called “two-hit models.” Commonly, early-life seizures do not result in the loss of neurons, although some studies have reported modest changes (compared with those produced in adults). Later in life, most models show increased susceptibility to seizure. In some models, a minority of animals become epileptic, developing spontaneous behavioral and/or electrographic seizures. Suggested underlying mechanisms vary from altered expression and/or function of several ion channels to changes in expression of the AMPAR subunit, GluR2 which may in turn diminish GABA_A receptor function. Another outcome from studies of early-life SE and brief/recurrent seizures are deficits in learning and memory. Dendritic abnormalities including spine loss may contribute to these behavioral deficits.

The field of experimental childhood Epilepsy Research has entered an era of great promise. Although many challenges remain, opportunities abound with respect to new animal models that are available—including emerging genetic mouse models—that should lead to a greater understanding of the molecular basis of these disorders and avenues for the development of new therapies.

Acknowledgments

The work of our laboratories was supported by: John W. Swann– NIH Grant NS18309, NIH Grant NS37171, and a grant from the Pediatric Partnership for Epilepsy Research; Tallie Z. Baram – NIH Grant NS28912, NIH Grant NS35439, NIH Grant R21 NS49618, and the American Epilepsy Society Research Initiative Fund; Frances E. Jensen – NIH Grant NS31718 and a grant from the Epilepsy Foundation; Solomon L. Moshe – NIH Grant NS20253 and a Heffer Family Research Grant. Dr. Moshe is also the recipient of the Martin A. and Emily L. Fisher Fellowship in Neurology and Pediatrics.

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	Epilepsy: A Comprehensive Textbook 2nd Edition
	© 2008 Lippincott Williams & Wilkins

Epilepsy: A Comprehensive Textbook2nd Edition

Epilepsy: A Comprehensive Textbook
2nd Edition