Profile of retigabine-induced neuronal apoptosis in the developing rat brain.

OBJECTIVE: Acute neonatal exposure to some, but not all, anticonvulsant drugs induces a profound increase in neuronal apoptosis in rats. Phenobarbital and phenytoin induce apoptosis at a therapeutically relevant dose range, lamotrigine and carbamazepine do so only at supratherapeutic doses or in polytherapy, and valproate does so even at subtherapeutic doses. Levetiracetam is devoid of pro-apoptotic effects. Retigabine, a new-generation drug, acts uniquely by enhancing the M-type potassium current. Because its safety profile in developing animals is unstudied, we sought to determine if retigabine would induce apoptosis. METHODS: Postnatal day (P) 7 rat pups were treated with retigabine (5-30 mg/kg), vehicle (saline), or comparator drugs (phenobarbital, lamotrigine, levetiracetam, or carbamazepine). Cell death was assessed using amino-cupric-silver staining. A separate group of animals was treated repeatedly (three times over 24 h) with retigabine (15 mg/kg) or vehicle. To establish a pharmacokinetic profile for retigabine, we measured plasma and brain levels after drug treatment. RESULTS: Consistent with prior studies from our group and others, we found phenobarbital-induced cell death throughout thalamus, nucleus accumbens, and several neocortical areas. By contrast, levetiracetam, lamotrigine, and carbamazepine were found to have no appreciable apoptotic effect on the aforementioned structures. Acute (single) exposure to retigabine, even at doses of 30 mg/kg, was also without effect on apoptosis. However, repeated (three times) exposure to retigabine triggered apoptosis in a subset of brain areas. The half-life of retigabine in plasma was 2.5 h, with appreciable concentrations reached in the brain within 1 h of administration. SIGNIFICANCE: These data demonstrate that retigabine, like many other anticonvulsant drugs, is capable of triggering neuronal apoptosis in the developing rat brain. Unlike other drugs, repeated dosing of retigabine was necessary to induce this effect. This may be due to its shorter half-life as compared to other drugs, such as phenobarbital.

Comparison of the long-term behavioral effects of neonatal exposure to retigabine or phenobarbital in rats.

Anticonvulsant drugs, when given during vulnerable periods of brain development, can have long-lasting consequences on nervous system function. In rats, the second postnatal week approximately corresponds to the late third trimester of gestation/early infancy in humans. Exposure to phenobarbital during this period has been associated with deficits in learning and memory, anxiety-like behavior, and social behavior, among other domains. Phenobarbital is the most common anticonvulsant drug used in neonatology. Several other drugs, such as lamotrigine, phenytoin, and clonazepam, have also been reported to trigger behavioral changes. A new generation anticonvulsant drug, retigabine, has not previously been evaluated for long-term effects on behavior. Retigabine acts as an activator of KCNQ channels, a mechanism that is unique among anticonvulsants. Here, we examined the effects retigabine exposure from postnatal day (P)7 to P14 on behavior in adult rats. We compared these effects with those produced by phenobarbital (as a positive control) and saline (as a negative control). Motor behavior was assessed by using the open field and rotarod, anxiety-like behavior by the open field, elevated plus maze, and light-dark transition task, and learning/memory by the passive avoidance task; social interactions were assessed in same-treatment pairs, and nociceptive sensitivity was assessed via the tail-flick assay. Motor behavior was unaltered by exposure to either drug. We found that retigabine exposure and phenobarbital exposure both induced increased anxiety-like behavior in adult animals. Phenobarbital, but not retigabine, exposure impaired learning and memory. These drugs also differed in their effects on social behavior, with retigabine-exposed animals displaying greater social interaction than phenobarbital-exposed animals. These results indicate that neonatal retigabine induces a subset of behavioral alterations previously described for other anticonvulsant drugs and extend our knowledge of drug-induced behavioral teratogenesis to a new mechanism of anticonvulsant action.

Brief postnatal exposure to phenobarbital impairs passive avoidance learning and sensorimotor gating in rats.

Phenobarbital is the most commonly utilized drug for the treatment of neonatal seizures. However, mounting preclinical evidence suggests that even brief exposure to phenobarbital in the neonatal period can induce neuronal apoptosis, alterations in synaptic development, and long-lasting changes in behavioral functions. In the present report, we treated neonatal rat pups with phenobarbital and evaluated behavior in adulthood. Pups were treated initially with a loading dose (80 mg/kg) on postnatal day (P)7 and with a lower dose (40 mg/kg) on P8 and P9. We examined sensorimotor gating (prepulse inhibition), passive avoidance, and conditioned place preference for cocaine when the animals reached adulthood. Consistent with our previous reports, we found that three days of neonatal exposure to phenobarbital significantly impaired prepulse inhibition compared with vehicle-exposed control animals. Using a step-though passive avoidance paradigm, we found that animals exposed to phenobarbital as neonates and tested as adults showed significant deficits in passive avoidance retention compared with matched controls, indicating impairment in associative memory and/or recall. Finally, we examined place preference conditioning in response to cocaine. Phenobarbital exposure did not alter the normal conditioned place preference associated with cocaine exposure. Our findings expand the profile of behavioral toxicity induced by phenobarbital.

Susceptibility to bystander DNA damage is influenced by replication and transcriptional activity.

Direct cellular DNA damage may lead to genome destabilization in unexposed, bystander, cells sharing the same milieu with directly damaged cells by means of the bystander effect. One proposed mechanism involves double strand break (DSB) formation in S phase cells at sites of single strand lesions in the DNA of replication complexes, which has a more open structure compared with neighboring DNA. The DNA in transcription complexes also has a more open structure, and hence may be susceptible to bystander DSB formation from single strand lesions. To examine whether transcription predisposes non-replicating cells to bystander effect-induced DNA DSBs, we examined two types of primary cells that exhibit high levels of transcription in the absence of replication, rat neurons and human lymphocytes. We found that non-replicating bystander cells with high transcription rates exhibited substantial levels of DNA DSBs, as monitored by γ-H2AX foci formation. Additionally, as reported in proliferating cells, TGF-ß and NO were found to mimic bystander effects in cell populations lacking DNA synthesis. These results indicate that cell vulnerability to bystander DSB damage may result from transcription as well as replication. The findings offer insights into which tissues may be vulnerable to bystander genomic destabilization in vivo.

Phosphorylation of histone H2A.X as an early marker of neuronal endangerment following seizures in the adult rat brain.

The phosphorylated form of histone H2A.X (γ-H2AX) is a well documented early, sensitive, and selective marker of DNA double-strand breaks (DSBs). Previously, we found that excessive glutamatergic activity increased γ-H2AX in neurons in vitro. Here, we evaluated γ-H2AX formation in the adult rat brain following neuronal excitation evoked by seizure activity in vivo. We found that brief, repeated electroconvulsive shock (ECS)-induced seizures (three individual seizures within 60 min) did not trigger an increase γ-H2AX immunostaining. In contrast, a cluster of 5-7 individual seizures evoked by kainic acid (KA) rapidly (within 30 min) induced γ-H2AX in multiple neuronal populations in hippocampus and entorhinal cortex. This duration of seizure activity is well below threshold for induction of neuronal cell death, indicating that the γ-H2AX increase occurs in response to sublethal insults. Moreover, an increase in γ-H2AX was seen in dentate granule cells, which are resistant to cell death caused by KA-evoked seizures. With as little as a 5 min duration of status epilepticus (SE), γ-H2AX increased in CA1, CA3, and entorhinal cortex to a greater extent than that observed after the clusters of individual seizures, with still greater increases after 120 min of SE. Our findings provide the first direct demonstration that DNA DSB damage occurs in vivo in the brain following seizures. Furthermore, we found that the γ-H2AX increase caused by 120 min of SE was prevented by neuroprotective preconditioning with ECS-evoked seizures. This demonstrates that DNA DSB damage is an especially sensitive indicator of neuronal endangerment and that it is responsive to neuroprotective intervention.

Effects of neonatal antiepileptic drug exposure on cognitive, emotional, and motor function in adult rats.

Despite the potent proapoptotic effect of several antiepileptic drugs (AEDs) in developmental rodent models, little is known about the long-term impact of exposure during brain development. Clinically, this is of growing concern. To determine the behavioral consequences of such exposure, we examined phenobarbital, phenytoin, and lamotrigine for their effects on adult behaviors after administration to neonatal rats throughout the second postnatal week. AED treatment from postnatal days 7 to 13 resulted in adult deficits in spatial learning in the Morris water maze and decreased social exploration for all drugs tested. Phenobarbital exposure led to deficits in cued fear conditioning, risk assessment in the elevated plus maze, and sensorimotor gating as measured by prepulse inhibition, but it did not affect motor coordination on the rotorod task. In contrast, phenytoin and lamotrigine exposure led to impaired rotorod performance, but no deficits in sensorimotor gating. Phenytoin, but not lamotrigine or phenobarbital, increased exploration in the open field. Phenytoin and phenobarbital, but not lamotrigine, disrupted cued fear conditioning. These results indicate that AED administration during a limited sensitive postnatal period is sufficient to cause a range of behavioral deficits later in life, and the specific profile of behavioral deficits varies across drugs. The differences in the long-term outcomes associated with the three AEDs examined are not predicted by either the mechanism of AED action or the proapoptotic effect of the drugs. Our findings suggest that a history of AED therapy during development must be considered as a variable when assessing later-life cognitive and psychiatric outcomes.

Early postnatal exposure of rats to lamotrigine, but not phenytoin, reduces seizure threshold in adulthood.

In view of previous reports of changes in seizure susceptibility in adult rats exposed to phenobarbital or diazepam as pups, we examined the effects of early life exposure to lamotrigine and phenytoin, two commonly used antiepileptic drugs (AEDs), for their effect on seizure threshold in adult rats. We found that pups exposed to lamotrigine for 6 days during the second postnatal week had a significantly lower threshold for pentylenetetrazole-evoked seizures when tested as adults. In contrast, phenytoin exposure during the second postnatal week was without a significant effect on seizure threshold in adults. Seizure scores at threshold were comparable across all groups tested. The dose of lamotrigine used in our study (20 mg/kg) was below that required to cause developmental neuronal apoptosis, whereas the dose of phenytoin used (50 mg/kg) was above that required for developmental neurotoxicity. Therefore, our findings suggest that neurodevelopmental alterations in seizure susceptibility may occur via mechanisms that are independent of those responsible for neural injury or teratogenesis. Our findings support the possibility that therapy with certain AEDs during pregnancy or infancy may alter seizure susceptibility later in life, a possibility that should be taken into account when examining early life factors that contribute to seizure susceptibility in adulthood.

Pattern of antiepileptic drug-induced cell death in limbic regions of the neonatal rat brain.

The induction of neuronal apoptosis throughout many regions of the developing rat brain by phenobarbital and phenytoin, two drugs commonly used for the treatment of neonatal seizures, has been well documented. However, several limbic regions have not been included in previous analyses. Because drug-induced damage to limbic brain regions in infancy could contribute to emotional and psychiatric sequelae, it is critical to determine the extent to which these regions are vulnerable to developmental neurotoxicity. To evaluate the impact of antiepileptic drug (AED) exposure on limbic nuclei, we treated postnatal day 7 rat pups with phenobarbital, phenytoin, carbamazepine, or vehicle, and examined nucleus accumbens, septum, amygdala, piriform cortex, and frontal cortex for cell death. Histologic sections were processed using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay to label apoptotic cells. Nucleus accumbens displayed the highest level of baseline cell death (vehicle group), as well as the greatest net increase in cell death following phenobarbital or phenytoin. Phenobarbital exposure resulted in a significant increase in cell death in all brain regions, whereas phenytoin exposure increased cell death only in the nucleus accumbens. Carbamazepine was without effect on cell death in any brain region analyzed, suggesting that the neurotoxicity observed is not an inherent feature of AED action. Our findings demonstrate pronounced cell death in several important regions of the rat limbic system following neonatal administration of phenobarbital, the first-line treatment for neonatal seizures in humans. These findings raise the possibility that AED exposure in infancy may contribute to adverse neuropsychiatric outcomes later in life.

Latency to onset of status epilepticus determines molecular mechanisms of seizure-induced cell death.

The molecular mechanisms mediating degeneration in response to neuronal insults, including damage evoked by prolonged seizure activity, show substantial variability across laboratories and injury models. Here we investigate the extent to which the proportion of cell death occurring by apoptotic vs. necrotic mechanisms may be shifted by changing the temporal parameters of the insult. In initial studies with continuous seizures (status epilepticus, SE), signs of apoptotic degeneration were most clearly observed when SE occurred following a long latency (>86 min) after injection of kainic acid as compared with a short-latency SE (<76 min). Therefore, in this study we directly compared short- with long-latency SE for the expression of molecular markers for apoptosis and necrosis in an especially vulnerable brain region (rhinal cortex). Molecular markers of apoptosis (DNA fragmentation, cleavage of ICAD, an inhibitor of "caspase-activated DNase" (CAD), and prevalence of a caspase-generated fragment of alpha-spectrin) were detected following long-latency SE. Short-latency SE resulted in expression of predominantly necrotic features of cell death, such as "non-ladder" pattern of genomic DNA degradation, prevalence of a calpain-generated alpha-spectrin fragment, and absence of ICAD cleavage. Silver staining revealed no significant difference in the extent and spatial distribution of degeneration between long- or short-latency SE. These data indicate that the latency to onset of SE determines the extent to which apoptotic or necrotic mechanisms contribute to the degeneration following SE. The presence of a long latency period, during which multiple brief seizure episodes may occur, favors the occurrence of apoptotic cell death. It is possible that the absence of such "preconditioning" period in short-latency SE favors predominantly necrotic profile.

Status epilepticus leads to the degradation of the endogenous inhibitor of caspase-activated DNase in rats.

Specific biochemical hallmarks of apoptosis, namely internucleosomal DNA fragmentation and caspase-3 activation, appear in the aftermath of status epilepticus (SE). This led us to hypothesize that caspase-activated DNase (CAD) is involved in DNA fragmentation and apoptotic neuronal cell death following SE. The present study aimed to determine whether SE is associated with an activation of CAD, as reflected in the degradation of the CAD inhibitor, ICAD. SE was induced in adult male Sprague-Dawley rats by kainic acid (12 mg/kg i.p.) and seizures were terminated with diazepam after 2 h. At 24, 48, or 72 h after SE termination, protein levels of CAD and ICAD were measured by Western blotting (after sodium dodecyl sulfate-polyacrylamide gel electrophoresis) using specific antibodies. At 48 and 72 h after SE termination, ICAD protein levels significantly decreased (by more than 60%) in rhinal cortex and hippocampus as compared with those in the same tissue from animals not experiencing SE. No changes were detected in total CAD protein levels at any time point, resulting in an increase in the ratio of CAD to its inhibitor. The loss of ICAD following SE is indicative of a disinhibition of CAD, leading to DNA fragmentation. Consistent with this, we observed that the decrease in ICAD between 24 and 48 h was accompanied by a marked increase in DNA fragmentation. Our results support the proposal that CAD participates in caspase-3-mediated internucleosomal DNA fragmentation in the aftermath of SE.

Ontogenic profile of seizures evoked by the beta-carboline DMCM (methyl-6,7-dimethoxy-4-ethyl-ß-carboline-3-carboxylate) in rats.

The beta-carboline, methyl-6,7-dimethoxy-4-ethyl-ß-carboline-3-carboxylate (DMCM), is a potent chemoconvulsant. While it has been utilized in adult rodents, it has not been previously examined for effects across postnatal development. DMCM is a negative allosteric modulator of benzodiazepine-sensitive GABAA receptors, receptor subtypes that are particularly enriched in limbic brain regions. This raises the possibility that DMCM may be particularly effective at evoking forebrain seizures, which is a challenge in neonatal animals due to the relative immaturity of the forebrain seizure network. The ability to selectebrain seizures is desirable when screening for drugs to use in temporal lobe epilepsy, which is characterized by seizures within the forebrain (limbic) network. To determine the profile of DMCM action across development, we examined the dose-dependent ability of DMCM to induce seizures in rats at P7, P10, P13, P14, P21 and in adulthood. We found that the highest sensitivity to DMCM occurred in P10, P13, and P14 rats. The lowest sensitivity occurred in P21 rats. Neonatal (P7) and adult (P60+) rats displayed moderate sensitivity. With moderate (0.2-0.4 mg/kg) doses of DMCM, we were able to reliably evoke limbic motor seizures without tonic-clonic components in animals as young as P7. These data support the utility of DMCM in assessing seizure threshold during development and raise the possibility for future exploration of DMCM as an agent to screen anticonvulsant drugs during the postnatal period.

Melatonin potentiates the anticonvulsant action of phenobarbital in neonatal rats.

Phenobarbital is the most commonly utilized drug for neonatal seizures. However, questions regarding safety and efficacy of this drug make it particularly compelling to identify adjunct therapies that could boost therapeutic benefit. One potential adjunct therapy is melatonin. Melatonin is used clinically in neonatal and pediatric populations, and moreover, it exerts anticonvulsant actions in adult rats. However, it has not been previously evaluated for anticonvulsant effects in neonatal rats. Here, we tested the hypothesis that melatonin would exert anticonvulsant effects, either alone, or in combination with phenobarbital. Postnatal day (P)7 rats were treated with phenobarbital (0-40mg/kg) and/or melatonin (0-80mg/kg) prior to chemoconvulsant challenge with pentylenetetrazole (100mg/kg). We found that melatonin significantly potentiated the anticonvulsant efficacy of phenobarbital, but did not exert anticonvulsant effects on its own. These data provide additional evidence for the further examination of melatonin as an adjunct therapy in neonatal/pediatric epilepsy.

Anticonvulsant effect of retigabine during postnatal development in rats.

Retigabine is a new-generation antiepileptic drug that exerts therapeutic action through the activation of KCNQ channel dependent M-type potassium currents. While retigabine has been extensively studied in adult animals using a wide variety of seizure models, its effects in developing animals have not been examined. There has only been one previous report of retigabine efficacy in juvenile rats (Mazarati et al., 2008), which examined efficacy against kindled seizures and did not examine ages younger than postnatal day (P) 14. To determine the efficacy of retigabine during brain development we pretreated rats with retigabine (0-30 mg/kg) at three ages corresponding to the neonatal period through late childhood/early adolescence (i.e., P7, P14, or P25). Seizures were induced 30 min later using a chemoconvulsant (pentylenetetrazol, PTZ) model, which has been widely used to determine anticonvulsant efficacy of many other antiepileptic drugs in neonatal animals. In a dose and age-dependent manner, retigabine reduced the severity of PTZ evoked seizures, increased the latency to seizure onset, and decreased the incidence of full maximal seizures. The minimum effective dose was found to be 5mg/kg for P7 animals, 2.5mg/kg for P14 animals, and 1mg/kg for P25 animals. These findings allow a direct comparison between retigabine and previously studied antiepileptic drugs against PTZ seizures during development, and provide the first report of the effective dose range of retigabine in neonatal animals.

Effects of repeated minimal electroshock seizures on NGF, BDNF and FGF-2 protein in the rat brain during postnatal development.

Repeated brief seizures, such as those induced by electroconvulsive therapy (ECT), markedly elevate neurotrophic factor levels in the adult rat brain, but it is not known whether a similar response to seizures occurs in immature animals. To address this question, we evoked brief seizures with electroconvulsive shock (ECS) in rat pups at different stages of postnatal development and examined basic fibroblast growth factor (FGF-2), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF) proteins in selected brain regions in which these trophic factors are known to increase in the adult rat following ECS-induced seizures. ECS treatments were administered daily (3 episodes/day) over 7 days to rat pups of three different ages: postnatal day (P)1-7, P7-13, or P14-20. Protein levels were measured 6h after the last ECS using Western blotting for FGF-2 in rhinal cortex, ELISA for BDNF and NGF in hippocampus, and NGF in frontal cortex. 7 days of repeated ECS-induced seizures during P1-7 did not alter protein levels for BDNF, FGF-2, or NGF. The repeated seizures during P7-13 affected only BDNF protein, causing a significant elevation of 40% in hippocampus over sham-treated controls. In P14-20 pups, the repeated seizures resulted in a significant increase in BDNF in hippocampus (162% over controls) and FGF-2 in rhinal cortex (34% over controls), while NGF protein did not show a significant change in either hippocampus or frontal cortex. The results suggest that during the first postnatal week there is a resistance to seizure-induced increase in neurotrophic factors, but by the third postnatal week, both BDNF and FGF-2 are elevated substantially in response to repeated seizures. This time-dependent profile suggests that synthesis of these proteins is initially activity-independent, becoming subject to activity-dependent regulation by 3 weeks of age. This maturation of seizure-evoked changes in trophic factors may be important for understanding the impact of ECT and seizures in childhood.

Epigenetic regulation of caspase-3 gene expression in rat brain development.

The expression levels of caspase-3, a major contributor to the execution of neuronal apoptosis, markedly decrease in the process of brain maturation. We have previously cloned the rat caspase-3 gene promoter and identified its essential regulatory elements. In the present study, we extended previous findings by examining transcriptional regulation of caspase-3 expression in the rat brain of two different ages, corresponding to the immature and mature brain. In particular, we determined that the rate of transcription initiation substantially declines during brain maturation. Furthermore, we established that mRNA levels of Ets1, Ets2, and Sp1 do not change in the brain with maturation, suggesting that these transcription factors do not contribute to age-dependent caspase-3 down-regulation. Hence, we examined a role of DNA methylation and histone modification in this process. Utilizing bisulfite DNA sequencing, we determined the presence of age-dependent differentially methylated fragments within the caspase-3 promoter region. Strikingly, differentially methylated CpG sites correspond to the predicted binding sites for a number of transcription factors that have been previously shown to be involved in neuronal development and differentiation. Moreover, using chromatin immunoprecipitation, we found that mature brains displayed significantly lower levels of histone 3 acetylated Lys14 and histone 4 acetylated Lys5, 8, 12, and 16. This observation is consistent with the decreased level of expression of caspase-3 in the mature brain. Together with our observation that histone deacetylase inhibitor, trichostatin A, increased the level of caspase-3 mRNA in cortical neurons in vitro, these results further indicate an important role of epigenetic factors in the regulation of caspase-3 gene expression.

Immunohistochemical evaluation of the protein expression of nerve growth factor and its TrkA receptor in rat limbic regions following electroshock seizures.

Repeated (but not acute) exposure to brief, non-injurious seizures evoked by minimal electroconvulsive shock (ECS) decreases neuronal death in limbic system and increases mRNA levels for nerve growth factor (NGF). Thus, the induction of NGF is a potential mechanism for the neuroprotection evoked by repeated ECS. The neuroprotective action of NGF is mediated by the TrkA receptor. This study determined whether repeated ECS exposure increased TrkA and NGF protein levels. To determine the functional significance of changes in these proteins, we compared the effects of ECS given daily either for 7 days (chronic ECS) or for 1 day (acute ECS). After chronic ECS, upregulation of both NGF and TrkA was found in perirhinal cortex, thalamus, and amygdala. In hippocampus, TrkA was upregulated in CA2, CA3 and CA4. NGF increase in hippocampus was found in CA1 and dentate gyrus. In frontal cortex and substantia innominata, an increase in NGF (but not in TrkA) was found. In most brain regions, TrkA and NGF remained unchanged after acute ECS. Our results demonstrate that repeated exposure to ECS causes an upregulation of TrkA and NGF proteins in several limbic areas in which neuroprotective effects are observed suggesting that NGF contributes to ECS-evoked neuroprotection.

Antiepileptic drug-induced neuronal cell death in the immature brain: effects of carbamazepine, topiramate, and levetiracetam as monotherapy versus polytherapy.

The aim of this study was to test the potential neurotoxicity of three antiepileptic drugs (AEDs), carbamazepine (5H-dibenzepine-5-carboxamide), topiramate [2,3:4,5-bis-O-(1-methylethylidene)-beta-d-fructopyranose sulfamate], and levetiracetam [2-(2-oxopyrrolidin-1-yl)butanamide], in the developing rat brain, when given alone or in combinations. The extent of cell death induced by AEDs was measured in several brain regions of rat pups (postnatal day 8) by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay 24 h after drug treatment. Carbamazepine alone did not increase neurodegeneration when given in doses up to 50 mg/kg, but it induced significant cell death at 100 mg/kg. When combined with phenytoin, carbamazepine, 50 but not 25 mg/kg, significantly exacerbated phenytoin-induced cell death. Although topiramate (20-80 mg/kg) alone caused no neurodegeneration, all doses exacerbated phenytoin-induced neurodegeneration. Levetiracetam (250-1000 mg/kg) alone did not induce cell death, nor did it exacerbate phenytoin-induced neurodegeneration. Of the combinations examined, only that of levetiracetam (250 mg/kg) with carbamazepine (50 mg/kg) did not induce neurodegeneration. Our data underscore the importance of evaluating the safety of combinations of AEDs given during development and not merely extrapolating from the effects of exposure to single drugs. Although carbamazepine and topiramate alone did not induce neuronal death, both drugs exacerbated phenytoin-induced cell death. In contrast, because cotreatment with levetiracetam and carbamazepine did not enhance cell death in the developing brain, it may be possible to avoid proapoptotic effects, even in polytherapy, by choosing appropriate drugs. The latter drugs, as monotherapy or in combination, may be promising candidates for the treatment of women during pregnancy and for preterm and neonatal infants.

Effects of lamotrigine alone and in combination with MK-801, phenobarbital, or phenytoin on cell death in the neonatal rat brain.

The neonatal rat brain is vulnerable to neuronal apoptosis induced by antiepileptic drugs (AEDs), especially when given in combination. This study evaluated lamotrigine alone or in combination with phenobarbital, phenytoin, or the glutamate antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) for a proapoptotic action in the developing rat brain. Cell death was assessed in brain regions (striatum, thalamus, and cortical areas) of rat pups (postnatal day 8) by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, 24 h after acute drug treatment. Lamotrigine alone did not increase neuronal apoptosis when given in doses up to 50 mg/kg; a significant increase in cell death occurred after 100 mg/kg. Combination of 20 mg/kg lamotrigine with 0.5 mg/kg MK-801 or 75 mg/kg phenobarbital resulted in a significant increase in TUNEL-positive cells, compared with MK-801 or phenobarbital treatment alone. A similar enhancement of phenytoin-induced cell death occurred after 30 mg/kg lamotrigine. In contrast, 20 mg/kg lamotrigine significantly attenuated phenytoin-induced cell death. Lamotrigine at 10 mg/kg was without effect on apoptosis induced by phenytoin. Although the functional and clinical implications of AED-induced developmental neuronal apoptosis remain to be elucidated, our finding that lamotrigine alone is devoid of this effect makes this drug attractive as monotherapy for the treatment of women during pregnancy, and for preterm or neonatal infants. However, because AEDs are often introduced as add-on medication, careful selection of drug combinations and doses may be required to avoid developmental neurotoxicity when lamotrigine is used in polytherapy.

Neurodevelopmental impact of antiepileptic drugs and seizures in the immature brain.

Seizure incidence during the neonatal period is higher than any other period in the lifespan, yet we know little about this period in terms of the effect of seizures or of the drugs used in their treatment. The fact that several antiepileptic drugs (AEDs) induce pronounced apoptotic neuronal death in specific regions of the immature brain prompts a search for AEDs that may be devoid of this action. Furthermore, there is a clear need to find out if a history of seizures alters the proapoptotic action of the AEDs. Our studies are aimed at both of these issues. Phenytoin, valproate, phenobarbital, and MK801 each induced substantial regionally specific cell death, whereas levetiracetam even in high doses (up to 1,500 mg/kg) did not have this action. In view of our previously findings of neuroprotective actions of repeated seizures in the adult brain, we also examined repeated seizures for a possible antiapoptotic action in the infant rat. Rat pups were preexposed to electroshock seizures (ECS) for 3 days (age 5-7 days) before receiving MK801 on day 7. The effect of ECS, which was consistently a 30% decrease in MK801-induced programmed cell death (PCD), suggests that repeated seizures can exert an antiapoptotic action in the infant brain. In contrast, PCD induced by valproate was not attenuated by ECS preexposure, suggesting that valproate-induced PCD is mechanistically distinct from that induced by MK801 and may not be activity-dependent. Presently, we do not know if this neuroprotective effect of seizures is deleterious or beneficial. If the seizures also enhance the survival of neurons that are destined to undergo naturally occurring PCD, early childhood seizures may have deleterious effects by preventing this necessary component of normal development. While this effect of seizures might be counteracted by AEDs, the fact that several AEDs shift the PCD to the other extreme, and trigger excessive neuronal cell loss, raises concern about whether the drug therapy may be more detrimental than the seizures. In this context, it is encouraging that we have identified at least one AED that is devoid of a proapoptotic action in the infant brain, even in high doses. It is now important to evaluate the long-term consequences of the changes in PCD in infancy by examining behavioral outcomes and seizure susceptibility in the AED- and seizure-exposed neonates when they reach adulthood.

Rapid phosphorylation of histone H2A.X following ionotropic glutamate receptor activation.

Excessive activation of ionotropic glutamate receptors increases oxidative stress, contributing to the neuronal death observed following neurological insults such as ischemia and seizures. Post-translational histone modifications may be key mediators in the detection and repair of damage resulting from oxidative stress, including DNA damage, and may thus affect neuronal survival in the aftermath of insults characterized by excessive glutamate release. In non-neuronal cells, phosphorylation of histone variant H2A.X (termed gamma-H2AX) occurs rapidly following DNA double-strand breaks. We investigated gamma-H2AX formation in rat cortical neurons (days in vitro 14) following activation of N-methyl-D-aspartate (NMDA) or alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate glutamate receptors using fluorescent immunohistochemical techniques. Moreover, we evaluated the co-localization of gamma-H2AX 'foci' with Mre11, a double-strand break repair protein, to provide further evidence for the activation of this DNA damage response pathway. Here we show that minimally cytotoxic stimulation of ionotropic glutamate receptors was sufficient to evoke gamma-H2AX in neurons, and that NMDA-induced gamma-H2AX foci formation was attenuated by pretreatment with the antioxidant, Vitamin E, and the intracellular calcium chelator, BAPTA-AM. Moreover, a subset of gamma-H2AX foci co-localized with Mre11, indicating that at least a portion of gamma-H2AX foci is damage dependent. The extent of gamma-H2AX induction following glutamate receptor activation corresponded to the increases we observed following conventional DNA damaging agents [i.e. non-lethal doses of gamma-radiation (1 Gy) and hydrogen peroxide (10 microm)]. These data suggest that insults not necessarily resulting in neuronal death induce the DNA damage-evoked chromatin modification, gamma-H2AX, and implicate a role for histone alterations in determining neuronal vulnerability following neurological insults.