Seizures in the developing brain: Cellular and molecular mechanisms of neuronal damage, neurogenesis and cellular reorganization

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Neurochemistry International 52 (2008) 935–947 www.elsevier.com/locate/neuint

Review

Seizures in the developing brain: Cellular and molecular mechanisms of neuronal damage, neurogenesis and cellular reorganization Irma E. Holopainen * Department of Pharmacology, Drug Development and Therapeutics, and Medicity Research Laboratory, Institute of Biomedicine, University of Turku, Tykisto¨katu 6A, 4th Floor, FIN-20014 Turku, Finland Received 14 May 2007; received in revised form 29 October 2007; accepted 31 October 2007 Available online 17 November 2007

Abstract Epilepsy is a common neurological disorder that occurs more frequently in children than in adults. The extent that prolonged seizure activity, i.e. status epilepticus (SE), and repeated, brief seizures affect neuronal structure and function in both the immature and mature brain has been the subject of increasing clinical and experimental research. Earlier studies suggest that seizure-induced effects in the immature brain compared with the adult brain are different. This is manifested as differences in neuronal vulnerability, cellular and synaptic reorganization and regenerative processes. The focus of this review is first to give a short overview of currently used experimental models of epilepsy in immature rats, and then discuss more thoroughly seizure-induced acute and sub-acute cellular and molecular alterations, highlight the contribution of inflammatory-like reactions and intracellular cytoskeleton to the insult, and reveal changes in the structure and function of inhibitory GABAA and excitatory glutamate receptors. The role of seizure-activated reparative, plastic processes, synaptic remodelling, neurogenesis as well as the long-term consequences of seizures are briefly outlined. The main emphasis is put on studies carried out in experimental animals, and the focus of interest is the hippocampus, the brain area of great vulnerability in epilepsy. In vitro studies are discussed only to limited extent. Collectively, recent studies suggest that the deleterious effects of seizures may not solely be a consequence of neuronal damage and loss per se, but could be due to the fact that seizures interfere with the highly regulated developmental processes in the immature brain. # 2007 Elsevier Ltd. All rights reserved. Keywords: Seizures; Developing brain; Nerve cell death; Neurogenesis

1. Experimental models of epilepsy in the developing brain Studies in animal models have made important contributions to our understanding of seizure-related brain injury and identification of its extent, duration, and long-term consequences for behaviour, and learning and memory processes. The further advantage of animal models is that they enable us to clarify seizure-related, time-dependent changes at different levels, from genes to behaviour. Although extrapolation of conclusions from animal data to humans must be done with caution, the use of experimental animals of various development ages give us important clues about the evolution of seizure-induced brain damage and its possible consequences in humans.

* Tel.: +358 2 333 7018; fax: +358 2 333 7000. E-mail address: [email protected]. 0197-0186/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2007.10.021

1.1. Animal models The most commonly used animal in epilepsy research of developing brain is the rat. Rats are born in a premature state relative to human, which must be taken into account when interpreting the data obtained in rat studies. Thus, a 8–10-dayold, postnatal (P) rat corresponds to a full-term neonate, a P12– 18 rat to an infant/toddler, and a P25–38 rat to a peripubertal child (Haut et al., 2004). The administration of convulsant drugs, kainic acid (KA), an agonist of the KA type of glutamate receptors, and pilocarpine (PC), an agonist for cholinergic muscarinic receptors (Ben-Ari, 1985; Turski et al., 1986, 1989) represent the most widely used epilepsy models in immature rats. PC is generally combined with lithium (Li) pretreatment, which potentiates the epileptogenic effect of PC, and reduces its peripheral cholinergic side effects and animal mortality (Jope et al., 1986). Depending on the dose and route of administration, KA and LiPC induce in rodents repeated limbic seizures or generalized status

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epilepticus (SE), which in many aspects are supposed to replicate human temporal lobe epilepsy (TLE) (Ben-Ari, 1985). In addition, repeated volatile flurothyl (bis-2,2,2-triflurothyl ether) inhalation, and an intraperitoneal (i.p.) injection of pentylenetetrazol (PTZ), an agonist for the picrotoxin binding site of the GABAA receptor complex (Ramanjaneyulu and Ticku, 1984; Hansen et al., 2004) are used as models of generalized seizures (Holmes et al., 1998). Kindling, induced by a continuous electrical stimulation of the hippocampus or amygdala is also used to some extent in immature animals serving as a model for partial seizures with secondary generalization (for references see Morimoto et al., 2004). Table 1 shows the most frequently used convulsive compounds to induced seizures/SE, age of animals (from P7 up to P28), the most pronouncedly affected brain regions, and the type of neuronal damage detected either acutely, sub-acutely, or after a prolonged follow-up period. In addition to the dose and route of the convulsant given, the age of animals seems to be one of the main factors determining the extent of damage, and it also delineates the long-term deleterious consequences of SE (Sankar et al., 1998; Liu et al., 1999; Sanchez and Jensen, 2001; Mikati et al., 2003). Several factors, such as intrinsic neuronal properties, degree of

myelination, state of receptor and ion channel maturation, and functional maturation of synaptic contacts could contribute to age-dependent differences in seizure propagation, as well as to the seizure-induced acute and long-term deleterious effects. Selected topics of these questions are discussed in more detail below. 2. Acute and sub-acute cellular and molecular changes induced by seizures and SE in the hippocampus of developing rats In principle, seizure-induced effects in the brain can be regarded as a three-stage process, in which a rapid neuronal death induced by an excitotoxic effect of glutamate is the initial phase. This is followed by the second phase, during which other deleterious processes, such as activation of apoptosis and cytokine-activated inflammatory processes, and on the other hand, neuroprotective responses mediated, e.g. by trophic factors, are activated. The third, long-lasting stage is then characterized by changes in cellular connectivity, synaptic reorganization, and functional alterations in the hippocampal circuitry and extrahippocampal networks. One hallmark of this reorganization process, defined as epileptogenesis, is the

Table 1 Seizure models, areas affected, and molecular mechanisms of neuronal damage in the developing rats Age

Seizure model

Type of injury and areas affected

Source

P7 P7

KA (i.c.v.) KA (i.c.v.)

Dong et al. (2003) Humphrey et al. (2002)

P9

KA

Neuronal death (CA1 and CA3) Acute necrotic (CA3), late (P40–P75) progressive apoptotic CA1 and CA3 nerve cell death Some microglia activation (hippocampus)

P9

KA

P9

KA

P10

PTZ

P10–P15 P11–P23

Flurothyl (repeated daily) Flurothyl (repeated daily)

P12 P12

LiPC LiPC

P12

LiPC

P15 P15

LiPC KA

P16 P16 P21

KA Amygdala kindling KA

P14–P28

LiPC

Changes in neurofilament protein expression in the hippocampus Increase in high-molecular weight MAP2 expression, disturbed dendritic structure in CA1 and CA3 Transient nerve cell injury (cortex, hippocampus, thalamus, hypothalamus), decreased metabolic rates in most cortical, hippocampal, and sensory areas Increase in COX-2 expression No neuronal death, MF sprouting, widespread c-fos activation (in rats having 50 seizures) Neuronal injury (thalamus) Necrotic neuronal injury and activation of microglia (thalamus) Volume reduction as adult (hippocampus, amygdala, perirhinal cortex)(MRI study) Neuronal injury (thalamus, CA1) Microglia activation, increased GFAP expression (hippocampus, some in extrahippocampal regions), increase in GFAP expression Some CA3c neuronal loss, no synaptic rearrangement No neuronal loss, no mossy fiber sprouting Nerve cell damage (CA1, CA3, amygdala, entorhinal and perirhinal cortex, periventricular nuclei), activated microglia, increase in cytokine expression Depending on the age of rats, neuronal damage in CA1, DG granule cells, and amygdala

Rizzi et al. (2003) and Ravizza et al. (2005) Lopez-Picon et al. (2004) Jalava et al. (2007) Nehlig and Pereira de Vasconcelos (1996) Kim and Jang (2006) Liu et al. (1999) Wasterlain et al. (2002) Kubova´ et al. (2002) Nairisma¨gi et al. (2006) Wasterlain et al. (2002) Rizzi et al. (2003) and Ravizza et al. (2005) Haas et al. (2001) Haas et al. (2001) Rizzi et al. (2003) and Ravizza et al. (2005) Sankar et al. (1998)

Abbreviations: COX-2 = cyclo-oxygenenase-2; CPPS = chronic perforant pathway stimulation; DG = dentate gyrus; GFAP = glial fibrillary acidic; i.c.v. = intracerebroventircular; LiPC = lithium pilocarpine; KA = kainic acid; MF = mossy fiber; MRI = magnetic resonance imaging; P = postnatal; PTZ = pentylenetetrazolone. The drugs were given intraperitoneally (i.p.) unless otherwise stated.

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alteration in seizure susceptibility manifested as unprovoked, spontaneous seizures, which further favour the gradual progression of cellular and molecular changes finally leading to other long-term deleterious consequences, such as learning and memory impairment. Fig. 1 shows the major changes induced by SE as a function of time in the postnatal rat hippocampus. It should be emphasized that the list is not exclusive, and the onset and duration of such changes are approximated. 2.1. Mechanisms of nerve cell death in response to seizures In adult rats, seizure-induced hippocampal nerve cell damage and their ultimate death is considered to occur acutely through necrosis, later followed by apoptotic and autophagocytic processes, or to have features of all these mechanisms (for references see Yuan et al., 2003; Henshall and Simon, 2005). A number of earlier studies suggest that the immature brain is less susceptible to seizure-induced damage than the adult, mature brain, and depending on the model used, either no nerve cell death or minor insults have been detected in the hippocampus, amygdala, and temporal cortical regions of animals younger than 2 weeks of age (Nitecka et al., 1984; Cavalheiro et al., 1987; Sperber et al., 1991; Haas et al., 2001; Cilio et al., 2003) although some short- and long-term metabolic changes may occur in the more mature rats (Nehlig and Pereira de Vasconcelos, 1996; Dube et al., 2000). Moreover, even a series of 55 seizures induced by flurothyl during the first 12 days of life have not resulted in any detectable acute or late-onset (when studied at P60) nerve cell loss either in the hippocampus, or piriform and parietal cortical areas (Riviello

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et al., 2002). The precise molecular mechanisms of seizureinduced nerve cell damage and death in the immature brain are still poorly defined. Results of such studies, in which neuronal damage has been detected, suggest that the mechanisms involved are in principle similar to those activated in adult animals, but the distribution of damage seems to be strictly agespecific, at least in the hippocampus (for references see Haut et al., 2004). For example, in a LiPC model, SE-induced damage in the CA1 neurons was maximal in P14 and P21 rats, whereas only a few damaged neurons were detected in the CA3 region of P14 rats although this specific cell population was extensively damaged in P21 rats. The SE-induced CA1 nerve cell death at the early age seems to involve both necrosis as evidenced by eosinophilic cells, and apoptosis as shown by nuclear fragmentation in TUNEL staining, and also verified by an electron microscopy (EM) (Sankar et al., 1998). Also neuronal damage in the amygdala and dentate granule cells was age-specific, and the vulnerability progressively increased with the age (Sankar et al., 1998). The route of convulsant given should also be taken into consideration when interpreting and comparing the extent and mechanism of neuronal death. Consequently, the intracerebroventricular (i.c.v.) application of KA has resulted in more severe acute and even progressive damage than that given intraperitoneally (i.p.). For example, the i.c.v. application of KA in P7 rats has lead to a dose-dependent acute neuronal loss in the CA3 region, later followed by a transient upregulation of heat shock protein (HSP-70), positive TUNEL staining, increased glial fibrillary acidic protein (GFAP) expression, and typical features of apoptosis in the CA3 and CA1 neurons at the EM level (Montgomery et al., 1999; Humphrey et al., 2002;

Fig. 1. Acute, sub-acute, and chronic changes induced by SE in the immature rat hippocampus. Note that the onset and duration of the changes are approximated. The question mark indicates that the currently available information is still lacking to confirm this specific consequence. Abbreviation: d = day/days.

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Dong et al., 2003), whereas an i.p. injection of KA in P9 rats has provoked SE without any detectable neuronal damage in the hippocampus (Rizzi et al., 2003; Lopez-Picon et al., 2004). Also regions other than the hippocampus could be affected as shown in P12 rats, in which SE (LiPC, i.p.) resulted in acute (within 12 h), necrotic neuronal damage in the central and lateral segments of the mediodorsal thalamic nucleus (Kubova et al., 2001). Apoptotic markers, caspase-3 and cytochrome c, remained unchanged, but activated microglia occurred within the damaged area. Moreover, the activation of inflammatory processes by necrotic neuronal damage was suggested to contribute to the further nerve cell injury. 2.2. Activation of inflammatory-like processes Activation of inflammatory processes by seizures is currently considered to be an important contributor to nerve cell death in adult animals (Kunz and Oliw, 2001; Jung et al., 2006; Lee et al., 2007), but its role in the immature brain is still poorly defined. The pattern of glial activation and cytokine gene transcription induced by SE are age-dependent (Rizzi et al., 2003), and thus activation of this death process seems to be as well developmentally regulated. In accordance, weak microglia activation has occurred acutely after SE in the hippocampus of P9 rats, whereas in older rats (P15 and P21), strongly immunoreactive cells resembling reactive microglia appeared not only in the hippocampus, but also in the extrahippocampal areas (Ravizza et al., 2005). Moroever, GFAP and interleukin (IL)-1b mRNAs were upregulated in P15 rats, while in older rats (P21), the expression of all the cytokines studied, i.e. IL1b, IL-6, and tumor necrosis factor (TNF)-a were extensively augmented, and their synthesis preceded the appearance of degenerating neurons (Ravizza et al., 2005). The mechanism by which cytokines contribute to neuronal death is still poorly known. It has been suggested that, e.g. the deleterious effects of IL-1b may be related to a functional interaction between its receptor and the N-methyl-D-aspartate receptor (NMDA) leading to alterations in the NMDA receptor channel-gating properties in a way that favours Ca2+ influx (Ali and Salter, 2001). The cytokine-activated signal transduction involves the induction of the developmentally regulated inducible nitric oxide synthesis (iNOS) (Romer et al., 1996), and/or the cyclooxygenase (COX) pathway (Tocco et al., 1997). COX-1 is constitutively expressed, whereas a transient activity of COX-2, which catalyzes the metabolism of arachidonic acid (AA) to prostaglandins (PGs), is induced by the pro-inflammatory cytokines and growth factors (Yamagata et al., 1993; O’Banion, 1999). COX-2 is a rate-limiting step in the PG synthesis, and its rapid upregulation in response to seizures has been proposed to contribute to the SE-induced hippocampal CA3 cell damage in adult rats (Tu and Bazan, 2003; Kawaguchi et al., 2005). The role of iNOS and COX-2 in excitotoxic nerve cell damage was recently also shown in P9 rats, in which an injection of NMDA (37 nM) into the sensorimotor cortex resulted in maximal de novo induction of iNOS and COX-2 within 10 h in the damaged area (Acarin et al., 2002). At the cellular level, iNOS appeared

in infiltrated neutrophils and in ramified protoplasmic astrocytes, which were closely associated with blood vessels, whereas the COX-2 immunoreactivity was mainly localized in the reactive microglial and neuronal cells. Moreover, the maximal iNOS and COX-2 expression preceded neuronal death suggesting their contribution to the death process (Acarin et al., 2002). The constitutive expression of COX-2, which increases markedly in the hippocampal neurons between P7 and P14, and reaches adult levels by P21 (Tocco et al., 1997), may thus contribute age-dependently to the seizure-activated inflammation process and consequent neuronal damage. Further corroboration for the functional role of inflammation in neuronal damage in the developing rats comes from a recent study, in which an i.p. injection of KA in P21 rats caused a twophase inflammatory response. The initial rapid (within 30 min) response seemed to be KA receptor-mediated, hippocampusspecific, and resulted in extensive production of PGs, most pronouncedly those of PGD2, PGF2a, PGE2 (Yoshikawa et al., 2006). The initial upregulation phase was blocked by a pretreatment of rats with KA receptor antagonists, and a COX-2 selective inhibitor (all given i.c.v.). During the late phase, sustained production of several PGs occurred together with an increase in the COX-2 mRNA (peaking 3–6 h after KA) and protein (3 h up to 24 h) expression. The significance of COX-2 in the SE-induced nerve cell death was also shown in adult rats, in which the selective COX-2 inhibitor, refecoxib, ameliorated excitotoxic neuronal damage (Hewett et al., 2006), and attenuated the number of TUNEL-positive cells in the hippocampus (Kunz and Oliw, 2001). Moreover, pretreatment of P7 rats with celocoxib prior to the flurothyl-induced seizures has delayed the appearance of seizure signs, and attenuated COX-2 expression (Kim and Jang, 2006). The altered PG production in response to seizures can also affect neuronal function as shown in a recent study, in which PGE2 dynamically regulated membrane excitability, synaptic transmission, and plasticity in the CA1 pyramidal neurons (Chen and Bazan, 2005). The diversity of the PG production pathways, to which both COX-1 and COX-2 differentially contribute (Yoshikawa et al., 2006), and their detailed role in the seizure-related nerve cell damage during the ontogeny is a challenge for further research. Moreover, the proposed neuroprotective effects of drugs specifically targeted at this pathway need to be further clarified in immature animals. 2.3. The importance of intracellular cytoskeleton in excitotoxic nerve cell damage Seizures can also effect the dynamic stability of the intracellular neuronal cytoskeleton, which is essential for the normal physiological plasticity of nerve cells and their survival (for references see Sanchez et al., 2000; Rami, 2003). Consequently, any severe enough disturbance in this dynamic structure could alter intracellular trafficking, dendrite morphology, and contribute to nerve cell death (Faddis et al., 1997; Kim et al., 2002). As a key promoting factor for the intracellular cytoskeletal disturbance is thought to be the excessive intracellular Ca2+ loading, which occurs during seizure activity

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(Lynch and Guttmann, 2002), and leads, besides its other intracellular effects, to activation of the Ca2+-dependent protease family, the calpains, ubiquitously expressed in the brain (Siman et al., 1989; Chan and Mattson, 1999). As substrates for calpains serve cytoskeletal proteins, such as neurofilament (NF) protein and microtubule-associated protein-2 (MAP2) (Pant, 1988; Rami, 2003). NF proteins are localized in the nerve cell soma and axons (Trojanowski et al., 1986; Lee and Cleveland, 1996), whereas MAP2 is expressed exclusively in dendrites (variably also in cell soma) (Matus et al., 1981; Tucker, 1990). The expression of the three NF proteins, the high, medium, and low-molecular-weight isoforms, and the high (HMW) and the low-molecular-weight (LMW) MAP2 proteins are strictly developmentally regulated and heterogeneously distributed in various nerve cell populations of the rat hippocampus (Lopez-Picon et al., 2004; Jalava et al., 2007). The importance of the cytoskeleton as a buffering organelle in stress situations was recently documented in P9 rats, in which the KA-induced SE resulted in rapid, but transient enhanced expression of NF and HMW MAP2 proteins without any neuronal damage (as verified by Fluoro-Jade staining) in the hippocampus (Lopez-Picon et al., 2004; Jalava et al., 2007). Such transient enhanced expression does not occur in adult rats. On the contrary, degradation of cytoskeletal proteins, e.g. NF and MAP2, has been detected after ischemia and seizures followed by nerve cell death, most notably in the hippocampal CA1and CA3 subregions (Wang et al., 1994; Yang et al., 1995; Dawson and Hallenbeck, 1996; Li et al., 1998; Sanabria et al., 2002; Lopez-Picon et al., 2004). In addition to the transient enhanced expression of cytoskeletal proteins in response to SE, also an acute (within 3 h) but transient formation of dendritic varicosities occurred in the CA1 and CA3 regions of P9 rats (Jalava et al., 2007), in organotypic hippocampal slice cultures (OHCs) exposed to sublethal NMDA concentrations (1–10 mM, 5 min), and after hypoxic-ischemic brain injury in infant rats (Ikonomidou et al., 1989). These dendritic abnormalities do not necessarily precede neuronal death, and their formation can be blocked by selective NMDA and non-NMDA receptor antagonists (Park et al., 1996; Arias et al., 1997; Ikegaya et al., 2001) suggesting that such changes may represent an early sign of disorganization of the neuronal cytoskeleton with a concomitant activation of repair/reorganization and self-protective intracellular compensation processes in the surviving neurons (Faddis et al., 1997; Ikegaya et al., 2001; Jalava et al., 2007). Moreover, seizure-induced calpain activation may also contribute to the dendritic remodelling through changes in the cytoskeletal structure after a brief excitotoxic injury as shown to take place in vitro conditions (Faddis et al., 1997). These studies favour the idea that the structural integrity of cytoskeletal proteins is of major importance for neuronal survival, and any severe enough disturbance in this dynamic structure could promote nerve cell death (Kim et al., 2002; Rami et al., 2003). On the other hand, stimuli of lower intensity could activate plastic repair processes manifested as transient alterations in the expression and localization of cytoskeletal proteins, e.g. NF and MAP2, without any obvious neuronal

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death. Indeed, a recent study in OHCs suggests that there exists a link between the degradation of the cytoskeletal proteins by calpain proteases and extensive excitotoxic neuronal damage (Lopez-Picon et al., 2006). Moreover, calpain inhibition has been effective in protecting neurons from the excitotoxic damage in adult rats (Wu et al., 2004; Higuchi et al., 2005), and to some extent also in OHCs (prepared from P6 rats and cultured for 1 week) (Lopez-Picon et al., 2006). Thus inhibitors of calpain could serve as important target molecules to promote structural integrity of nerve cells, and enhance their viability after seizures. 3. Seizure-induced changes in the inhibitory GABAA and excitatory glutamate receptor systems The balanced activity of the inhibitory and excitatory neurotransmitter system in the brain is of essential importance for the normal brain function, and any disturbance in this genuine balance can lead to seizure activity. Table 2 summarises the results of recent studies, which show seizure-induced alterations in the inhibitory and excitatory receptor systems in different experimental models of epilepsy in the developing rat. 3.1. Changes in the GABAA receptor expression and function In the developing brain, the GABAA receptor function undergoes extensive and profound changes during the early postnatal phase, as activation of GABAA receptors leads to neuronal excitation in the developing brain, while hyperpolarizing the mature, adult neurons (Leinekugel et al., 1997; BenAri, 2001). This differences is mainly due to the maturational change in the transmembrane chloride gradient, which is temporally correlated with the expression of the neuronal K+/ Cl cotransporter KCC2, the protein responsible for extruding intracellular Cl from cells (Rivera et al., 1999). Also the structure of GABAA receptors undergoes extensive changes during the postnatal development, as the expression of several subunits alters during the postnatal period subunit-specifically in several brain regions, including the hippocampus (Laurie et al., 1992; Fritschy et al., 1998; Brooks-Kayal et al., 1998, 2001; Laure´n et al., 2005). For example, the expression of a1 subunit is low at birth, and it gradually increases with the maturation, while changes in the a2 subunit expression have the opposite trend (Laurie et al., 1992; Fritschy et al., 1994; Brooks-Kayal et al., 1998). The developmentally regulated maturation process can be disturbed by seizures as shown in P9 rats, in which SE resulted in region-selective alterations in the expression of a1, a2, b3, and g2 subunit mRNAs in the hippocampus, some of which persisted up to 1 week (Laure´n et al., 2005). Moreover, the altered subunit expression can lead to changes in the pharmacology of the GABAA receptor as recently shown to occur acutely and sub-acutely after SE in P9 rats (Laure´n et al., 2007). In these rats, the displacement of [3H]flunitrazepam (an agonist specific for the a1b2g2 receptor type) binding by zolpidem (the benzodiazepine type I specific ligand) decreased

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Table 2 Seizure-induced alterations in excitatory and inhibitory receptors and neurogenesis in the hippocampus of developing rats Age

Model of SE

Type of change

Reference

P0-P4 P5

Repeated flurothyl KA

McCabe et al. (2001) Sathanoori et al. (2004)

P6, P9, P13

KA single or x 3

P7 P9 P9

KA (i.c.v.) KA KA

P10

LiPC

P10 P12 P20

PTZ (seizures twice daily) KA LiPC

P20

LiPC

P20 P22

LiPC, KA KA

Reduced neurogenesis in DG and hilus Increase in BDNF exon III and exon IV transcripts in the CA3 region Elevation in mGluR1 a protein expression in a select group of interneurons of the CA1 stratum oriens Late phase neurogenesis (CA3 and granule cell layer) Region specific changes in GABAA receptor subunit expression Acute and sub-acute changes in GABAA receptor pharmacology in the hippocampus Long-term changes in GABAA receptor a1 subunit expression in the DG Increase in granule cell neurogenesis Down-regulation of KA receptors (revealed by binding assay) Increase in the number of survived and matured DG granule cells (followed from P21 up to P50) Different changes in AMPA and KA receptor subunits in mature and newly born immature granule cells GABAA receptor a1 subunit changes in DG granule cells Increased phospho-Trk and BDNF immunoreactivities in the MF pathway

Avallone et al. (2006) Dong et al. (2003) Laure´n et al. (2005) Laure´n et al. (2007) Zhang et al. (2004) Holmes et al. (1999) Tandon et al. (2002) Porter et al. (2004) Porter et al. (2006) Raol et al. (2006) Danzer et al. (2004)

Abbreviations: AMPA = BDNF = brain-derived neurotrophic factor; DG = dentate gyrus; i.c.v. = intracerebroventircular; KA = kainic acid; LiPC = lithium pilocarpine; MAP2 = microtubule-asssociated protein-2; MF = mossy fiber; P = postnatal; PTZ = pentylenetetrazolone; SE = status epilepticus. The drugs were given intraperitoneally (i.p.) unless otherwise stated.

sub-acutely in several hippocampal subregions (CA3 and CA1), and acutely in the dentate gyrys (DG), parietal cortex, and thalamus. This resulted in transient appearance of receptors with lower sensitivity for zolpidem (Laure´n et al., 2007). It is noteworthy that alterations in the mRNA and protein expression as well as in the binding properties occurred without any neuronal death (Laure´n et al., 2005, 2007). The altered benzodiazepine binding has also been detected in the DG, several cortical areas, certain amygdaloid nuclei, and substantia nigra 3 days after KA-induced SE in P14 rats (Rocha et al., 2000). In addition to these drastic acute and sub-acute effects of SE on the properties of GABAA receptors in young rats, SE can also lead to long-term changes in the receptor structure as shown in P20 rats, in which SE-induced alterations in the GABAA receptor a1 subunit mRNA and protein expression persisted up to 3 months in the DG, were not model specific, and preceded the onset of epilepsy (Raol et al., 2006). Alterations in the expression of subunits, and their altered incorporation pattern in the GABAA receptor complex can lead to long-term functional changes in the GABAA receptormediated inhibition, as shown in rats after SE at P10. These rats had long-term GABAA receptor changes including the two-fold increase in the a1 subunit mRNA expression, and enhanced benzodiazepine type I augmentation of the GABA-mediated currents in adulthood (Zhang et al., 2004). The possible dual (both excitatory and inhibitory) actions of GABAA receptor activation at the early age (La¨msa¨ et al., 2000; Khazipov et al., 2004) include the fact that seizures generated with the functional GABAergic synapses in the immature brain show fast oscillations that are required to transform the normal network activity to an epileptic one. Moreover, the inhibition of GABAA receptors can prevent long-lasting consequences of

seizures in the immature, but not in the adult brain (Khalilov et al., 2005). 3.2. Changes in the glutamate receptor expression and function Seizures can also affect the expression of excitatory receptor subunits, of which the metabotropic glutamate receptors (mGluRs) are of interest. These receptors are coupled to G proteins, and once activated, mediate slow synaptic responses, and play a role in synaptic plasticity, modulation of neuronal excitability, and neurotransmitter release (Conn and Pin, 1997). It has been shown that the expression of mRNA and protein of several mGluR subtypes is developmentally regulated in the hippocampus (Catania et al., 1994; Defagot et al., 2002), and analogously to the GABAA receptors, seizure-induced alterations have also been detected in the mGlu and ionotropic KA receptors. For example, recurrent KA-induced seizures in P12 rats are associated with the down-regulation of KA receptors in the CA3 and dentate regions as measured with the [3H]KA binding 4 days after SE (Tandon et al., 2002). Moreover, KAinduced SE has elevated the mGluR1a protein expression within a select group of inhibitory interneurons located at the CA1 stratum (str) oriens-alveus, amygdala, and piriform cortex (Avallone et al., 2006). This specific change was suggested to serve as a potential mechanism on how early-life seizures could synchronize limbic network suppression, prevent further propagation of seizures, and contribute to the resistance and tolerance of the immature hippocampus to damage (Avallone et al., 2006). Also the KA-induced calpain activation, and an early increase in the AMPA (a-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid) immunoreactivity in the pyramidal

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layer after seizures may indicate that the calpain-mediated regulation of AMPA receptors plays a role in some pathophysiological processes following seizure activity (Bi et al., 1997). Of the ionotrophic glutamate receptors, NMDA receptors play a crucial role in neuronal development, plasticity, and survival (Collingridge and Lester, 1989; Cull-Candy et al., 2001). In addition, the high Ca2+ permeability of NMDA receptors during prolonged activation is involved in the pathophysiology of seizure-related neuronal death (Dingledine et al., 1999; Guttmann et al., 2002). During the postnatal period, the composition of the NMDA receptors is changed (Riva et al., 1994; Ritter et al., 2002) leading to altered kinetics of the NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) (Barth and Malenka, 2001) and to developmental changes in the NMDA receptor-mediated toxicity (Zhou and Baudry, 2006). This suggests that developmental changes in the receptor structure may be of major importance regulating the NMDA receptor-mediated toxicity. It is also of interest to note that proteolysis of the NMDA receptor NR2B subunit by calpain has occurred in the adult rat hippocampus after SE in same areas in which calpain was activated (Araujo et al., 2005). Moreover, it seems that the susceptibility of the NMDA receptor to cleavage by calpain varies with neuronal maturity in a manner that may also alter its electrophysiological properties (Dong et al., 2006). Collectively, experimental animal studies suggest that seizure activity affects both the excitatory and inhibitory receptor systems by disturbing the normal, strictly developmentally regulated expression patterns of both the glutamate and GABAA receptor subunit mRNAs and protein resulting in altered pharmacological and functional properties of the receptors, and thus contribute to the reorganization of the hippocampal circuitry with enhanced seizure susceptibility. Moreover, it has been suggested that the synergistic action of GABAA and NMDA receptors are required to trigger the cascades involved in epileptogenesis of the developing, but not in the adult hippocampus (Khalilov et al., 2005). More detailed information is needed to understand these age-specific dynamic alterations in the receptor structure and function, and how seizures can disturb this inherent physiological process in the developing brain. 4. Seizure-induced plastic changes and altered neurogenesis in the developing brain In addition to the deleterious effects on cell survival, receptor structures and integrity of intracellular cytoskeleton, SE also induces plastic changes in nerve cells and effects neurogenesis, which can enhance the survival of nerve cell populations, but which can as well turn out to be epileptogenic. Thus a lack of nerve cell death after SE or repeated seizures do not exclude the possibility that other cellular and molecular alterations of functional importance could take place. 4.1. Seizure-induced hippocampal plasticity One of the plastic changes induced by seizure activity in the hippocampus is the laminar specific sprouting of CA3

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pyramidal cell axons, the mossy fibers (MFs), to the CA3 str pyramidale and inner molecular layers (IMLs) of the DG, the phenomenon frequently detected in adult rats after SE (Tauck and Nadler, 1985; Okazaki et al., 1995; Wuarin and Dudek, 1996), but invariably in immature animals. For example, repetitive, daily flurothyl-induced seizures in rats from P11 to P23, and an intrahippocampal (P7) and i.p. injection of KA (P16-P20) have resulted in MF sprouting in the IML and CA3 str pyramidale in adulthood (Leite et al., 1996; Liu et al., 1999; Cilio et al., 2003). Moreover, recurrent daily PTZ-induced seizures in rats as young as P0 have altered the MF targeting in the CA3 region, and twice daily induced seizures in P10 rats have resulted in the MF sprouting in the CA3 and IML regions (Holmes et al., 1999). Although the precise molecular mechanisms of seizureinduced plasticity is still poorly known, the effects mediated through growth factors could be one feasible possibility, since seizures enhance the brain-derived nerve growth factor (BDNF) mRNA expression in the hippocampus age-dependently in the immature and juvenile rats (Kornblum et al., 1997; Danzer et al., 2004). For example, the phospho-Trk, an indicator of the activated BDNF receptor, and the BDNF immunoreactivity were robustly augmented in the MF pathway 24 h after KAinduced SE in P22 rats, intermediate phospho-Trk levels were detected in P17 rats, but no change occurred in P8 rats (Danzer et al., 2004). Of interest is that the ontogeny of the SE-induced BDNF expression and TrkB activation seemed to correlate with the ontogeny of the epileptogenic effects in the LiPC and KA SE models. Moreover, spontaneous seizures occurred later in such rats, which had SE at the older age (P18 and P21), but not in those having SE at the younger age (P10–P14) (Stafstro¨m et al., 1992; Priel et al., 1996; Sankar et al., 1998, 2002). Based on the ontogeny of the BDNF immunoreactivity, TrkB activation, and SE-induced epileptogenesis, the role of BDNF could be of importance in contributing to the MF sprouting and activation of epileptogenic cascade, which finally leads to spontaneous, unprovoked seizures. It should, however, be emphasized that in addition to plastic changes in the hippocampus, seizures may also disturb the normal developmental maturation program in other brain regions as well. For example, rats subjected to consecutive SE episodes between P7 and P9 exhibited reduced neocortical apoptosis, and disorganization of the cortical developmental patterns manifested as abnormal cellular distribution, and aberrant circuits (Valotta da Silva et al., 2005). Although such plastic processes may have functional, long-term consequences, the mechanisms and molecules involved in the reorganization events have remained poorly defined, and it seems that nerve cell loss is not an absolute prerequisite for their occurrence. 4.2. Seizure-induced alteration in neurogenesis Seizures also induce alterations in the neurogenesis, which may have a long-term impact on the developing brain. Recently, recurrent flurothyl-induced seizures in P0 and P4 rats resulted in attenuated DG granule cell neurogenesis starting from 36 h

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after seizures, and lasted up to 2 weeks, whereas granule cell neurogenesis was enhanced in the adult rats under similar experimental conditions (McCabe et al., 2001) as well as in other epilepsy models of adult rats (Parent et al., 1997; Ekdahl et al., 2003). In P20 rats, not only the birth, but also the death of DG granule cells was increased after SE during the follow-up period (from day 1 up to 3 weeks), and the majority of new DG granule cells actually later disappeared (Porter et al., 2004). However, despite the fact that only a subset of newly proliferated granule cells may survive, it has been shown that in fact more newborn neurons than in control rats have matured, and integrated into the hippocampal circuitry (Kobayashi and Buckmaster, 2003). This new functional organization may indeed serve as a potential epileptogenic factor, which could further modify the hippocampal circuitry. The age of animals at the time of seizures may also determine whether neurogenesis is enhanced or attenuated. For example, three episodes of KA-induced SE within the first 13 postnatal days have dramatically suppressed neurogenesis of the DG granule cells (Liu et al., 2003). Since only about 20% of granule cells are present at birth, and about 50% by P5 in the rat (Bayer, 1980), early life seizures, when affecting the proliferation rate of granule cells, could have pronounced effects on the brain development. This is also corroborated by a recent study, which showed that the early postnatal hilar area in rats is a unique neurogenic region, where cell proliferation, neuronal differentiation, and cell migration occur in a strictly orderly- and developmentally-regulated manner (Namba et al., 2005). This again emphasizes the difference in seizure effects between the immature and adult brain, and underlies the fact that seizures can be detrimental even in the absence of cell loss. The increase in the generation of DG granule cells and concurrent alterations in the neurotransmitter receptor expression can both simultaneously contribute to the development of spontaneous seizures. For example, a recent study showed that 2 weeks after SE (at P20), the majority of changes in the AMPA and KA receptor subunit mRNAs, i.e. decrease in GluR3 and GluR6, and increase in GluR2 and KA2 occurred in the mature population of DG granule cells, whereas in the newly born, immature granule cells the GluR6 expression was decreased (Porter et al., 2006). Since the SE-induced changes in the excitatory receptors favoured the occurrence of such receptors, which had a several-fold increase in the conductance, i.e. the KA-GluR6 heteromeric receptors compared to the homomeric GluR6 receptors, they could contribute to the augmented KA receptor conductance, and altered DG granule cell excitability seen in chronic epilepsy (Epsztein et al., 2005). It should also be emphasized that some of the SE-induced changes can, however, be beneficial for the cells. For example, the increase in the GluR2 receptor subtype may lead to a decrease in Ca2+ permeability (Liu et al., 2004), and in fact attenuate the seizureinduced intracellular Ca2+ load thus ameliorating the excitotoxic neuronal damage. Although the precise molecular mechanisms of seizureinduced granule cell neurogenesis are still poorly known, some hypothetical mechanisms have been suggested. For example, the drugs effecting the proliferation rate of neurons after

seizures may also suppress the abnormal excitatory activity as recently shown in adult rats, in which a selective COX-2 inhibitor, celecoxib, prevented neuronal death and microglia activation in the hilar and CA1 regions, and inhibited the generation of new glial cells in the CA1 region and ectopic granule cells in the hilus (Jung et al., 2006). Moreover, agedependent differences in the pattern and magnitude of seizureinduced BDNF mRNA expression (Sathanoori et al., 2004) suggest that the lower BDNF mRNA expression, which was restricted to the CA3 region in the KA-treated pups, but in the DG region of the adult rats, may indeed be one common factor underlying the observed age-dependent differences in cell damage, reactive sprouting, and neurogenesis. 5. Long-term functional and structural consequences of seizures in the developing rats Currently, the best characterized long-term consequences of early-life seizures are those affecting the hippocampal plasticity, e.g. sprouting, and cognitive functions, specifically those affecting learning and memory. In animal studies, SE in P1–P14 rats has resulted in long-term impairment in the radial arm maze performance, a hippocampus-dependent spatial memory task (Lynch et al., 2000), and in the visuospatial learning and memory tests in adulthood in rats having SE at P16–P20 (Cilio et al., 2003). In both experiments, also the facilitation of subsequent kindling was detected in adulthood. It is, however, still unsettled whether or not abnormalities in the MF pathway, e.g. sprouting, are causally related to the longterm impairment in the hippocampus-dependent memory tasks (Holmes et al., 1998; Huang et al., 1999). At variance, a recent study indicated that the L-type Ca2+ channel blocker given in P14 mice (corresponds roughly to P16–P21 rats) prevented MF sprouting, and ameliorated cognitive deterioration when tested with the spatial and contextual tasks at the age of P39–P51 (Ikegaya et al., 2000). These changes occurred without any anticonvulsant effect against the LiPC-induced seizures. Also functions other than those mediated by the hippocampus can be affected. For example, the SE-induced necrotic damage in the thalamus in P12 rats suggests that functions, such as adaptation to novelty, might become compromised as adults (Kubova et al., 2001). New imaging techniques have been applied to further verify seizure-induced long-term effects in the brain structure. A recent MRI study indicated that SE in P12 rats can lead to a reduction of hippocampal volume in a subset of animals, when they reach adulthood (Nairisma¨gi et al., 2006). This study suggested that although SE as early as at P12 can induce neurodegeneration (acute neuronal damage in the hippocampus, amygdala and perirhinal cortex), factors other than neuronal death could contribute to the development of brainvolume reduction and epileptogenesis after the early-life SE. Fig. 2 summarizes the current knowledge of the most important short- and long-term consequences of SE in the immature and mature brain. As discussed earlier, a part of consequences are clearly age-dependent, whereas in particular the long-term consequences are still poorly verified in the immature brain.

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Fig. 2. The main short- and long-term consequences of SE in the immature and mature rat hippocampus based on the information currently available. Note that some of the short-term consequences in the immature brain are strictly age-dependent, and the long-term consequences are still inadequately verified in the immature brain. Abbreviation: DG = dentate gyrus.

Although conclusions drawn from animal studies must be done with caution taking into account the age and model used, the results, however, emphasize the long-term deleterious effects of SE on cognitive processes, and altered seizure susceptibility in adulthood, and strongly suggest that similar alterations can take place during the course of an epileptic process in children. 6. Concluding remarks The age-dependency of seizure-induced, region-specific nerve cell death, alterations in the dynamic intracellular cytoskeletal structure, and changes at the receptor level create the basis for synaptic reorganization leading ultimately to functional changes in the hippocampal circuitry as well as in other brain structures functionally connected to the hippocampus. The precise cellular mechanisms of the altered seizure susceptibility after early-life seizures, and other long-term deleterious consequences as adults are, however, still poorly defined. Collectively, earlier results corroborate the idea that

this process includes numerous sequential, simultaneous, and over-lapping both deleterious and plastic, regenerative alterations, and may reflect seizure-promoting changes in the circuitries previously activated, and not merely be signs of a general increase in the neuronal excitability. It is also becoming more evident that the devastating effects of the early-age frequent and prolonged seizures may be primarily due to their interference with the highly regulated developmental processes rather than neuronal damage and loss per se, and this may occur not only in the hippocampus, but also in the extrahippocampal regions (Ben-Ari and Holmes, 2006). Also alterations in neurogenesis, and the extent to which the newly generated cells are integrated into the hippocampal network play an essential role in the modulation of functional activity. Our knowledge of the cellular and molecular pathways that regulate nerve cell death, and the complex reorganization process after seizures in the developing brain is at its early stages. Much more effort is required to elucidate these developmentally regulated processes to approach the goals

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of improving the treatment of epilepsy in children with the target- and age-specific drugs. This review provides not only up-to-date information of some of the processes involved in the complex reorganization cascade activated by seizures, but the aim is also to highlight the importance of the developing brain as a unique, dynamic structure within the field of neurochemistry and epilepsy research, and to awaken the interest for further new, innovative ways to approach this fascinating research field. Acknowledgements The financial support of the Academy of Finland (projects 8118471 and 8117438), and the Arvo and Lea Ylppo¨ Foundation is gratefully acknowledged. The technical assistance of Hanna Laure´n, M.Sc. with the cartoons is appreciated. References Acarin, L., Peluffo, H., Gonzalez, B., Castellano, B., 2002. Expression of inducible nitric oxide synthase and cyclooxygenase-2 after excitotoxic damage to the immature rat brain. J. Neurosci. Res. 68, 745–754. Ali, D.W., Salter, M.W., 2001. NMDA receptor regulation by Src kinase signalling in excitatory synaptic transmission and plasticity. Curr. Opin. Neurobiol. 11, 336–342. Araujo, I.M., Xapelli, S., Gil, J.M., Mohapel, P., Petersen, A., Pinheiro, P.S., Malva, J.O., Bahr, B.A., Brundin, P., Carvalho, C.M., 2005. Proteolysis of NR2B by calpain in the hippocampus of epileptic rats. Neuroreport 16, 393– 396. Arias, C., Arrieta, I., Massieu, L., Tapia, R., 1997. Neuronal damage and MAP2 changes induced by the glutamate transport inhibitor dihydrokainate and by kainate in rat hippocampus in vivo. Exp. Brain Res. 116, 467–476. Avallone, J., Gashi, E., Magrys, B., Friedman, L.K., 2006. Distinct regulation of metabotropic glutamate receptor (mGluR1 alpha) in the developing limbic system following multiple early-life seizures. Exp. Neurol. 202, 100–111. Barth, A.L., Malenka, R.C., 2001. NMDAR EPSC kinetics do not regulate the critical period for LTP at thalamocortical synapses. Nat. Neurosci. 4, 235– 236. Bayer, S.A., 1980. Development of the hippocampal region in the rat. II. Morphogenesis during embryonic and early postnatal life. J. Comp. Neurol. 190, 115–134. Ben-Ari, Y., 1985. Limbic seizure and brain damage produced by kainic acid: mechanisms and relevance to human temporal lobe epilepsy. Neuroscience 14, 375–403. Ben-Ari, Y., 2001. Developing networks play a similar melody. Trends Neurosci. 24, 353–360. Ben-Ari, Y., Holmes, G.L., 2006. Effects of seizures on developmental processes in the immature brain. Lancet Neurol. 5, 1055–1063. Bi, X., Chen, J., Baudry, M., 1997. Developmental changes in calpain activity, GluR1 receptors and in the effect of kainic acid treatment in rat brain. Neuroscience 81, 1123–1135. Brooks-Kayal, A.R., Jin, H., Price, M., Dichter, M.A., 1998. Developmental expression of GABA(A) receptor subunit mRNAs in individual hippocampal neurons in vitro and in vivo. J. Neurochem. 70, 1017–1028. Brooks-Kayal, A.R., Shumate, M.D., Jin, H., Rikhter, T.Y., Kelly, M.E., Coulter, D.A., 2001. Gamma-aminobutyric acid(A) receptor subunit expression predicts functional changes in hippocampal dentate granule cells during postnatal development. J. Neurochem. 77, 1266–1278. Catania, M.V., Landwehrmeyer, G.B., Testa, C.M., Standaert, D.G., Penney Jr., J.B., Young, A.B., 1994. Metabotropic glutamate receptors are differentially regulated during development. Neuroscience 61, 481–495. Cavalheiro, E.A., Silva, D.F., Turski, W.A., Calderazzo-Filho, L.S., Bortolotto, Z.A., Turski, L., 1987. The susceptibility of rats to pilocarpine-induced seizures is age-dependent. Brain Res. 465, 43–58.

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