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)-␤-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.

Summary Phenobarbital and phenytoin, two drugs commonly used for the treatment of neonatal seizures, have been well-documented to induce neuronal apoptosis throughout many regions of the developing rat brain. 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 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. Histological sections were processed using the 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, while 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.

Summary: 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.

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 (ϩ)-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 phenytoininduced cell death occurred after 30 mg/kg lamotrigine. In contrast, 20 mg/kg lamotrigine significantly attenuated phenytoininduced 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.

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.