Seizure Mechanisms and Vulnerability in the Developing Brain

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.

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
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
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

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