Abstract  Previous studies have demonstrated that recurrent seizures during the neonatal period lead to permanent changes in seizure threshold and learning and memory. The pathophysiological mechanisms for these changes are not clear. To determine if neonatal seizures cause changes in hippocampal excitability or inhibition, we subjected rats to 50 flurothyl-induced seizures during the first 10 days of life (five seizures per day). When the rats were adults, we examined seizure threshold using flurothyl inhalation, and learning and memory in the water maze. In separate groups of animals, we evaluated in vivo paired-pulse facilitation and inhibition in either CA1 with stimulation of the Schaffer collaterals or dentate gyrus with stimulation of the perforant path. Following these studies, the animals were sacrificed and the brains evaluated for mossy fiber sprouting with the Timm stain. Compared to control animals, rats with 50 flurothyl seizures had a reduced seizure threshold, impaired learning and memory in the water maze, and sprouting of mossy fibers in the CA3 pyramidal cell layer and molecular layer of the dentate gyrus. No significant differences in impaired paired-pulse inhibition was noted between the flurothyl-treated and control rats. This study demonstrates that recurrent neonatal seizures result in changes of neuronal connectivity and alterations in seizure susceptibility, learning and memory. However, the degree of impairment following 50 seizures was modest, demonstrating that the immature brain is remarkably resilient to seizure-induced damage.

Abstract  PURPOSE: Our previous research indicated that the exposure of rat pups to an hypoxic environment during a discrete developmental period (postnatal days 10-15) produces short-term seizures and confers an enduring increase in susceptibility to pentylenetetrazol- and flurothyl-induced seizures. In this study, we evaluated the effects of hypoxic insult in this neonatal period of susceptibility to electrical kindling and corneal electroconvulsive shock.

METHODS: Ten-day-old rat pups were exposed to a 3% O2 environment, as previously described, and were either kindled or exposed to corneal electroshock at adulthood (70 days old).

RESULTS: Neither kindled seizure development from the septal nucleus or amygdala nor electroconvulsive shock profiles were significantly altered by hypoxic pretreatment.

CONCLUSIONS: Results indicate that hypoxia produces increases in seizure susceptibility that are observable in only some experimental seizure models but not in others. This outcome serves to target some anatomic systems more than others in the mechanisms involved in hypoxia-induced neural reorganization.

Abstract  An epileptic seizure and postictal period in addition to well-known features are also characterized by massive consumption of energy. This is thought to lead to oxidative stress and increased generation of free radicals, which is reflected by increased levels of oxidative products. Our previous work described the neuroprotective effects of melatonin in preventing cognitive worsening after a single epileptic seizure. This work was aimed on direct measurement of free radicals in brain tissue using the EPR method 1, 15 and 60 minutes after seizure. The measurement was performed in adult male Wistar rats at the mentioned intervals after a single tonic-clonic seizure induced by flurothyl. In comparison to control animals there was a significant increase in hydroxyl and nitroxyl radicals 60 minutes after the seizure. The levels of hydroxyl radicals were significantly lower in animals that received melatonin 60 minutes before seizure induction compared to animals without preventive treatment. Therefore, melatonin affected the generation of the measured free radicals differently. An important finding was the delayed increase in free radicals after a single seizure in the later phases of recovery.

Abstract  Voltage-gated sodium channels are required for the initiation and propagation of action potentials. Mutations in the neuronal voltage-gated sodium channel SCN1A are associated with a growing number of disorders including generalized epilepsy with febrile seizures plus (GEFS+),(7) severe myoclonic epilepsy of infancy, and familial hemiplegic migraine. To gain insight into the effect of SCN1A mutations on neuronal excitability, we introduced the human GEFS+ mutation SCN1A-R1648H into the orthologous mouse gene. Scn1a(RH/RH) mice homozygous for the R1648H mutation exhibit spontaneous generalized seizures and premature death between P16 and P26, whereas Scn1a(RH/+) heterozygous mice exhibit infrequent spontaneous generalized seizures, reduced threshold and accelerated propagation of febrile seizures, and decreased threshold to flurothyl-induced seizures. Inhibitory cortical interneurons from P5-P15 Scn1a(RH/+) and Scn1a(RH/RH) mice demonstrated slower recovery from inactivation, greater use-dependent inactivation, and reduced action potential firing compared with wild-type cells. Excitatory cortical pyramidal neurons were mostly unaffected. These results suggest that this SCN1A mutation predominantly impairs sodium channel activity in interneurons, leading to decreased inhibition. Decreased inhibition may be a common mechanism underlying clinically distinct SCN1A-derived disorders.

Abstract  The consequences of epileptic seizures related to postictal inhibition in early postictal period include postictal analgesia. We studied this phenomenon over 96 h following flurothyl-induced seizures in adult male Wistar rats. Nociception of control (no seizure) and seizured groups were tested using the plantar and von Frey hair tests. We determined latency of forepaw and hind paw reactions using plantar tests and the number of von Frey hairs reactions. Shortly after seizures, longer plantar test latencies were seen relative to the control group. Before the seizures the plantar test reaction times were significantly shorter in forepaws than in hind paws. The effect disappeared post-seizure and surprisingly, it also disappeared at the corresponding time in controls; it reappeared after 48 h in the seizure group and after 24 h in controls. Differences in the von Frey hairs test occurred at 5 and 60 min post-seizure, however, these differences could not be explained by limb anatomy; although, different thermal and mechanical nociception mechanisms could be significant. The unexpected reactions in controls could be related to brief social and physical interactions between the two groups.

Abstract  The aim of the present study was to establish a recurrent convulsion model during the developmental stage using inhalation of flurothyl, and to observe the relationship between the changes in mitochondrial function in hippocampal neurons and hippocampal neuronal apoptosis after recurrent convulsion. A total of 36 Sprague-Dawley male rats were selected and randomly divided into the control (NS) group and recurrent-seizure (RS) group for 0, 1.5, 3, 12 and 24 h. After the last seizure the rats were subdivided with 6 animals in each group. Rats in the seizure group inhaled flurothyl repeatedly to induce status convulsivus, 30 min once daily, for 7 consecutive days, while the same operation was conducted in the control group without inhalation of flurothyl. At each time-point after the last seizure, blood was taken from the heart, followed by decapitation and immediate removal of the brain. Half of the brain tissue was immediately fixed in 10% paraformaldehyde to prepare paraffin-embedded tissues for hematoxylin and eosin (H&E) histological staining. Hippocampus was taken from the other half of the brain and stored at -80°C. Changes in mitochondrial membrane potential (ΔΨm) in hippocampal neurons were detected by flow cytometer. Dynamic changes of mitochondrial fusion and division-related genes, mitochondrial fusion protein 2 (Mfn2) and dynamin-related protein 1 (Drp1), in the hippocampus after recurrent convulsion were observed using reverse transcription-polymerase chain reaction (RT-PCR)and western blot analysis. The expression of caspase-3 and cytochrome c (Cyt c) was determined by RT-PCR and western blot analysis. After successful establishment of the recurrent convulsion model in rats during developmental stage using flurothyl, H&E staining results exhibited that in the CA1 region of hippocampus in the NS group, karyopyknosis occurred in nucleus that was stained to be brown and yellow, and the expression peak of apoptotic cells mainly existed at 24 h after the last convulsion. RT-PCR and western analysis revealed that apoptosis-related gene caspase-3 expression in the RS group was elevated at 1.5 h after the last convulsion, and lasted 24 h after convulsion. Detection results of mitochondrial ΔΨm revealed a significant reduction 1.5, 3 and 12 h after convulsion in hippocampal neurons of experimental rats, which reached the trough at 12 h, and rapidly increased after 24 h. The expression of Mfn2 mRNA in the RS group was significantly lower than that in the control group, while the expression of Drp1 mRNA in RS group was distinctly higher than that in the control group. RT-PCR and western blot analysis revealed that, mitochondrial apoptosis-related gene Cyt c expression was increased at 3 h after the last convulsion, and lasted 24 h after convulsion. Correlation analysis showed that the changes in mitochondrial function were closely related to neuronal apoptosis. The results of the study show that apoptosis exists in the hippocampus of rats after recurrent convulsion, which is closely related to the changes in mitochondrial function.

Abstract  Febrile seizure (FS) remains the most common childhood neurological emergency. Although many studies have been done, controversy exists as to whether these seizures are associated with a significant risk for cognitive impairment. The aim of our study is to check whether there is a spatial learning and memory deficit in the experimental FS rats using a heated-air FS paradigm and to determine the possible molecular mechanism of cognitive impairment. On days 10 to 12 postpartum, the male rat pups were subjected to one, three, or nine episodes of brief hyperthermia-induced seizures (HS). At adolescence and adulthood, the rats subjected to three, or nine episodes of HS had significant deficits in spatial learning and memory tested by Morris water maze. At adulthood, no apparent hippocampal neuronal loss was found in any HS group, but the seizure threshold to flurothyl was decreased significantly in the rats subjected to nine episodes of HS. In the rats subjected to three, or nine episodes of HS, the Western immunoblotting showed that there was a significant translocation of Ca(2+)-calmodulin stimulated protein kinase II (CaMKII) from the postsynaptic density to the cytosol. In the postsynaptic density the phosphorylation of CaMKIIα Thr(286) was reduced significantly, but the phosphorylation of CaMKIIα Thr(305) was increased significantly. Our study showed early-life brief but recurrent HS caused long-term cognitive impairment and CaMKIIα was involved in carrying forward the signal resulting from HS. The change of the phosphorylative level in Thr(286) and Thr(305) sites of CaMKIIα may underlie the molecular mechanism for the HS related cognitive impairment.

Abstract  Endogenous PGE(2) dynamically regulates membrane excitability, synaptic transmission and plasticity. Neonatal seizures are associated with a number of activity-dependent changes in brain development including altered synaptogenesis and synaptic plasticity as well as reduction in neurogenesis. Thus, it is reasonable to hypothesize that alteration of cyclooxygenase-2 (COX-2) expression induced by neonatal seizure may influence brain development. We evaluated the expression of COX-2 and microsomal prostaglandin E synthase (mPGES) by Western blot analysis and immnohistochemistry in flurothyl-induced neonatal seizure and also studied the effect of celecoxib on seizure induction. Seven to 10 days old Sprague-Dawley rats were used for control (n = 18) and experimental group (n = 30). Recurrent seizure group showed more increased level of COX-2 expression than control group. However, the level of mPGES-2 expression was similar in both groups, and mPGES-1 was not detected. Hippocampus of control rats showed endogenous COX-2 expression, which was localized mainly in CA3 region. This localization pattern was similar in recurrent seizure rats, but intensity of COX-2 expression was more increased than in control rats. Celecoxib treatment significantly delayed the seizure attack and also reduced COX-2 expression. In conclusion, this study suggests that COX-2 expression is related to epileptogenesis in flurothyl-induced neonatal seizure model and shows the possibility that its inhibition lessens functional impairments that occurred in neonatal seizure.

Abstract  Status epilepticus and repeated seizures have age-dependent morphological and neurophysiological alterations in the hippocampus. In the present study, effects of flurothyl-induced status epilepticus were examined in awake and free moving immature (2 weeks old) and adult rats. Without exception, adult rats died of respiratory arrest before the onset of status epilepticus. We were unable to find a concentration of flurothyl that produced status epilepticus and a low mortality in adult rats. In contrast, immature rats survived flurothyl status epilepticus for up to 60 min with a very low mortality. In rat pups, behavioral manifestations correlated with electrographic seizures in both the cortex and hippocampus. Neuropathological damage (cell loss, pyknotic cells or gliosis) was not observed in the immature hippocampus, thalamus, amygdala, substantia nigra or cortex at 24 h, 2 days or 2 weeks after status epilepticus. In addition, no aberrant mossy fiber reorganization or decrease in cells counts were observed in the hippocampus. Young rats did not show alterations in paired-pulse perforant path inhibition following flurothyl status epilepticus. The present findings are consistent with studies in other seizure models, indicating that immature rats are highly resistant to seizure-induced changes.

Abstract  Autonomic changes accompany seizures in both animals and humans. While ictal autonomic dysfunction can be life-threatening, the participating neural networks involved are poorly understood. In this study we examined the activation of Fos following generalized seizures in brainstem structures known to mediate autonomic function. Adult female rats were sacrificed 2 h after flurothyl-induced seizures. Double-immunostaining for c-Fos and dopamine-beta-hydroxylase (DBH), and c-Fos and phenylethanol-N-methyl-transferase (PNMT) were performed in brainstem slices. Numbers of DBH-labeled neurons expressing Fos-like immunoreactivity (FLI) (DBH/Fos) and PNMT labeled neurons expressing FLI (PNMT/Fos) were counted in the noradrenergic (A1, A2, A5, A7) and adrenergic (C1, C2) cell groups localized in pons and medulla oblongata. Among the experimental animals, the highest degree of co-localization of DBH/Fos neurons was observed in the locus coeruleus (A6; 87.7%), and in the A1(72.8%) cell group located in the caudal ventrolateral medulla (VLM). No co-localization of DBH/Fos neurons was observed in control animals. The highest degree of co-localization of PNMT/Fos neurons was observed in the C1 adrenergic cell group (84.2%) located in the rostral VLM. Control animals showed very few (5.5%) PNMT/Fos co-localized neurons in the C1 adrenergic cell group. Our results indicate that flurothyl-induced generalized seizures in rats activate catecholaminergic neurons in the pons and medulla oblongata. Further studies are necessary to determine whether activation of brainstem catecholaminergic neurons contribute to the autonomic manifestations that frequently accompany epileptic seizures.

Abstract  BACKGROUND: Although seizures are very common in neonates and are often the harbinger of poor neurologic outcome, there is controversy regarding the degree of brain damage induced by seizures during early development. Here, we evaluated the effect of neonatal seizures on subsequent brain injury induced by status epilepticus.

METHODS: Twenty-five seizures were induced by the inhalant flurothyl in neonatal rats during the first 5 days of life. Flurothyl reliably produced generalized seizures with concomitant electroencephalographic changes and a low mortality rate. During adolescence or early adulthood, animals were subjected to status epilepticus using either kainic acid or perforant path stimulation.

RESULTS: Although flurothyl-induced neonatal seizures did not cause cell death, animals that had neonatal seizures had significantly more severe brain injury after both kainic acid and perforant path stimulation than did animals without a history of neonatal seizures.

CONCLUSIONS: Neonatal seizures increase the susceptibility of the developing brain to subsequent seizure-induced injury.

Abstract  The Kv2.1 delayed rectifier potassium channel exhibits high-level expression in both principal and inhibitory neurons throughout the central nervous system, including prominent expression in hippocampal neurons. Studies of in vitro preparations suggest that Kv2.1 is a key yet conditional regulator of intrinsic neuronal excitability, mediated by changes in Kv2.1 expression, localization and function via activity-dependent regulation of Kv2.1 phosphorylation. Here we identify neurological and behavioral deficits in mutant (Kv2.1(-/-) ) mice lacking this channel. Kv2.1(-/-) mice have grossly normal characteristics. No impairment in vision or motor coordination was apparent, although Kv2.1(-/-) mice exhibit reduced body weight. The anatomic structure and expression of related Kv channels in the brains of Kv2.1(-/-) mice appear unchanged. Delayed rectifier potassium current is diminished in hippocampal neurons cultured from Kv2.1(-/-) animals. Field recordings from hippocampal slices of Kv2.1(-/-) mice reveal hyperexcitability in response to the convulsant bicuculline, and epileptiform activity in response to stimulation. In Kv2.1(-/-) mice, long-term potentiation at the Schaffer collateral - CA1 synapse is decreased. Kv2.1(-/-) mice are strikingly hyperactive, and exhibit defects in spatial learning, failing to improve performance in a Morris Water Maze task. Kv2.1(-/-) mice are hypersensitive to the effects of the convulsants flurothyl and pilocarpine, consistent with a role for Kv2.1 as a conditional suppressor of neuronal activity. Although not prone to spontaneous seizures, Kv2.1(-/-) mice exhibit accelerated seizure progression. Together, these findings suggest homeostatic suppression of elevated neuronal activity by Kv2.1 plays a central role in regulating neuronal network function.

Abstract  The cholinergic system modulates cerebral excitability. We recently reported that immunolesions of the basal forebrain (BF) cholinergic neurons in adult rats increase the susceptibility to generalized seizures. In this study we investigated the effects of lesions of the BF cholinergic neurons in neonatal rats on seizure susceptibility and cognitive function. Neonatal rats at postnatal day (P) 7 received intracerebroventricular (i.c.v.) injections of 192 IgG-saporin (SAP) or phosphate-buffered saline. Following 3 weeks after the injection the first group of rats was implanted with hippocampal electrodes for electroencephalogram (EEG) recordings while the second group of rats was tested for visual spatial memory using the hidden platform version of the water maze test. The first group of rats was then tested for seizure susceptibility using flurothyl 1 week after the electrode implantation. Rats that received immunolesions of the BF cholinergic neurons at P7 had significantly shorter latencies to onset of myoclonic jerks and tonic-clonic seizures than controls. However, no significant differences were found in the duration of seizures, or EEG ictal duration. No significant deficits in spatial learning were found between rats that received i.c.v. injections of SAP at P7 and controls. As in adult rats, lesions of the BF cholinergic system in rat pups result in subsequent increase in seizure susceptibility.

Abstract  There is increasing evidence that seizures during early development can impact ultrasonic vocalizations (USVs) emitted from neonatal mice. However, most of the effects of early-life seizures have been reported using chemoconvulsants that produce continuous seizures (status epilepticus). In the present study, we evaluated the impact of different seizure frequency loads during early-life vocalization development in C57BL/6J male and female mice. For the high seizure load (HSL) paradigm, we administered 3 flurothyl seizures to mice on postnatal day (PD) 7 through PD11, and recorded USVs on PD12. We found that the induction of seizures across PD7-11 resulted in increased average duration (P < 0.05) and cumulative duration (P < 0.05) of USVs across both sexes. Call-type analyses indicated several call-type changes, including reduced production of complex call-types from males' HSL condition. For the low seizure load (LSL) paradigm, we induced 3 flurothyl seizures only on PD10 and recorded USVs on PD12. We found no change in any spectral or temporal features of USVs. However, call-type production analyses indicated that both male and female animals from the LSL paradigm also produced changes in call-types. This study provides evidence that the magnitude of communication impairment following seizures is significantly impacted by seizure frequency load early in development.

Abstract  Perinatal hypoxia is associated with both seizures arising acutely and the subsequent development of temporal lobe epilepsy (as determined retrospectively). We therefore attempted to identify acute and chronic morphological and/or electrophysiological hippocampal pathologies associated with experimentally induced hypoxia in immature rats. Pups were exposed to 15 minutes of hypoxia on 3 successive days (starting on postnatal day 8; P8), or to 60 minutes of hypoxia on P10 with either one or multiple hypoxia-induced seizures. For animals experiencing multiple seizures, flurothyl seizure threshold was significantly lower than that of controls at 60 to 80 days, but not at 10 days, after hypoxia. Acutely, there was a treatment-related increase in the number and the density of pyknotic dentate and hilar neurons, in particular in animals experiencing multiple seizures. However, 60 to 80 days after the multiple-seizure protocol, the number of dentate and hilar neurons did not differ between control and experimental animals. Electrophysiological measures of pyramidal cell properties showed no striking difference between experimental and control animals at any time point. These results indicate that early postnatal hypoxia and hypoxia-induced seizure episodes decrease seizure threshold in the adult but produce minimal acute or chronic morphological or functional changes in the hippocampus.

Abstract  Interleukin-33 (IL-33) is a novel identified chromatin-associated cytokine of IL-1 family cytokines. It signals through a heterodimer comprised of ST2L and IL-1RAcp, and plays a crucial role in many diseases. However, very little is known about the role and underlying intricate mechanisms of IL-33 in recurrent neonatal seizure (RNS). To determine whether IL-33 plays an important regulatory role, we established a neonatal seizure model in this study. Rats were subjected to recurrent seizures induced by inhaling volatile flurothyl. Recombinant IL-33 or PBS were also administered by intraperitoneally (IP) before surgery, respectively. Here, our current results indicated that RNS contributed to a significant reduction in IL-33 and its specific receptor (ST2L) expressions in cortex. While, in hippocampus, RNS induced an increase in IL-33 and ST2L evidently, compared with Sham group. After injection with IL-33, however, a remarkable increase in total IL-33 was detected both in brain cortex and hippocampus. In addition, IL-33 was mainly co-localized in the nuclear of GFAP+ astrocytes and the cytoplasm of the Iba-1+ microglia and IL-33+/NeuN+ merged cells. In parallel, ST2L was expressed mainly in the membrane of GFAP+ astrocytes, Iba-1+ microglia and NeuN+ neurons, respectively. Furthermore, administration of IL-33 improved RNS-induced behavioral deficits, promoted bodyweight gain, and ameliorated spatial learning and memory ability. Moreover, IL-33 pretreatment blocked the activation of NF-κB, resisted inflammatory cytokines IL-1β and TNF-α increase, as well as suppressed apoptosis and autophagy activation after RNS. Collectively, IL-33 provides potential neuroprotection through suppressing apoptosis, autophagy and at least in part by NF-κB-mediated inflammatory pathways after RNS.

Abstract  There is considerable evidence that thalamic nuclei are involved in the propagation and regulation of seizures. In the present study, we investigated the possible role in seizure mechanisms of GABAergic transmission in two thalamic nuclei, the posterior nucleus (PO) and the ventromedial nucleus (VM). Several GABAergic drugs were bilaterally microinfused into PO or VM of adult rats via chronically implanted cannulae, before testing the rats' susceptibility to seizures induced by flurothyl. In PO, infusions of the GABA elevating agent gamma-vinyl-GABA (20 micrograms) or of the GABAA receptor agonist muscimol (100 ng) suppressed both clonic and tonic seizures. Infusions into PO of the GABAA receptor antagonist bicuculline (100 ng) facilitated both these seizure types. Administration of the GABAB receptor agonist baclofen (200 ng) also suppressed clonic seizure susceptibility. Drug infusions into VM, however, did not significantly modify the susceptibility to seizures. These findings lead us to conclude that GABAergic transmission in the vicinity of the PO, but perhaps not in VM, affects flurothyl seizure susceptibility. We hypothesize that GABA synapses in PO may be part of a seizure propagation or control circuit including striatum, substantia nigra, and superior colliculus.

Abstract  The aim of this study was to determine whether the regional distribution and time course of immunoreactivity to the c-fos protein varies with maturation and method of seizure induction. The effect of the two chemical convulsants, pentylenetetrazol (PTZ) and flurothyl, on the spatial and temporal pattern of c-fos-like immunoreactivity in immature (postnatal day (P) 10) was compared to that in adult rats. Patterns of c-fos-like immunoreactivity following O2 deprivation were also evaluated at the 2 ages because hypoxia is acutely epileptogenic in immature animals but not adults. C-fos-like immunoreactivity was examined at 2, 4, and 6 h after onset of chemically induced seizures or O2 deprivation at both ages. After PTZ or flurothyl seizures, both ages exhibited similar patterns of IR in amygdala, pyriform cortex, and hypothalamus. Age-dependent regional differences were most prominent in cortex: superficial layers of retrosplenial, cingulate, and neocortex stained in adults; staining was confined to deep layers of neocortex in P10 rats. Intense staining of dentate gyrus and hippocampus occurred with more prolonged seizures, but not brief seizures. PTZ administration resulted in staining at 2 h after seizure onset and was reduced by 4 h in adults, but immunoreactivity was not seen until 4 and 6 h after seizure onset in immature rats, indicating an age effect on the time course of IR. In immature rats, immunoreactivity patterns after hypoxia were markedly different from PTZ or flurothyl: staining was confined to layer VI of neocortex in these animals, and rarely involved limbic structures. These differences in the pattern of c-fos immunoreactivity suggest that the neuronal populations involved in epileptogenesis are influenced by age as well as seizure phenotype and intensity.

Abstract  Seizures in preterm infants are associated with a high risk of neurological sequelae. In the neonatal rat recurrent seizures have been associated with long-term changes in cerebral excitability and cognition as well as sprouting of mossy fiber terminals in the granule cell layer of the dentate gyrus and hippocampal CA3 subfield. To evaluate the relationship between seizure-induced morphological changes and cognitive function we subjected newborn rats to 55 seizures with flurothyl during the first 12 days of life. During adolescence rats with prior recurrent seizures were compared with controls in electroencephalographic power and performance in the Morris water maze and open field test. Rats subjected to recurrent seizures had marked impairment in water maze performance and never reached the level of learning seen in controls despite a total of 54 trials. Recurrent seizures were also associated with an overall reduction in spectral power which was most pronounced in the theta range. On histological examination rats with recurrent neonatal seizures had sprouting of mossy fiber terminals in CA3 and the granule cell layer of the dentate gyrus without any accompanying cell loss. Sprouting in CA3, but not the granule cell layer of the dentate gyrus, correlated with water maze performance. This study demonstrates that recurrent neonatal seizures can result in profound impairment of water maze performance and reduction of electroencephalographic power despite the lack of discernible cell loss and that this cognitive impairment correlates with mossy fiber sprouting in CA3.

Abstract  PURPOSE: There is a gap in our knowledge of the factors that modulate the predisposition to seizures following perinatal hypoxia. Herein, we investigate in a mouse model the effects of two distinct factors: developmental stage after the occurrence of the perinatal insult, and the presence of a seizure predisposing mutation.

METHODS: Effects of age: P6 (postnatal day 6) mouse pups were subjected to acute hypoxia down to 4% O2 over the course of 45 min. Seizure susceptibilities to flurothyl-induced seizures (single exposures) and to flurothyl kindling were determined at specific subsequent ages. Effects of mutation: Heterozygote mice, with deletion of one copy of the Kcn1a gene, subjected to P6 hypoxia were compared as adults to wild-type mice with respect to susceptibility to a single exposure to flurothyl and to the occurrence of spontaneous seizures as detected by hippocampal electroencephalography (EEG) and video recordings.

KEY FINDINGS: Effects of age: As compared to controls, wild-type mice exposed to P6 hypoxia had a shortened seizure latency in response to a single flurothyl exposure at P50, but not at P7 or P28 (p < 0.04). In addition, perinatal hypoxia at P6 enhanced the rate of development of flurothyl kindling performed at P28-38 (p < 0.03), but not at P7-17. Effects of mutation: Kcn1a heterozygous mice subjected to P6 hypoxia exhibited increased susceptibility to flurothyl-induced seizures at P50 as compared to Normoxia heterozygote littermates, and to wild-type Hypoxia and Normoxia mice. In addition, heterozygotes exposed to P6 hypoxia were the only group in which spontaneous seizures were detected during the period of long-term monitoring (p < 0.027 in all comparisons).

SIGNIFICANCE: Our data establish a mouse model of mild perinatal hypoxia in which we document the following: (1) the emergence, after a latent period, of increased susceptibility to flurothyl-induced seizures, and to flurothyl induced kindling; and (2) an additive effect of a gene mutation to the seizure predisposing consequences of perinatal hypoxia, thereby demonstrating that a modifier (or susceptibility) gene can exacerbate the long-term consequences of hypoxic injury.

Abstract  The ketogenic diet (KD) has been shown to be effective as an antiepileptic therapy in adults, but it has not been extensively tested for its efficacy in neonatal seizure-induced brain damage. We have previously shown altered expression of zinc/lipid metabolism-related genes in hippocampus following penicillin-induced developmental model of epilepsy. In this study, we further investigated the effect of KD on the neurobehavioral and cognitive deficits, as well as if KD has any influence in the activity of zinc/lipid transporters such as zinc transporter 3 (ZnT-3), MT-3, ApoE, ApoJ (clusterin), and ACAT-1 activities in neonatal rats submitted to flurothyl-induced recurrent seizures. Postnatal day 9 (P9), 48 Sprague-Dawley rats were randomly assigned to two groups: flurothyl-induced recurrent seizure group (EXP) and control group (CONT). On P28, they were further randomly divided into the seizure group without ketogenic diet (EXP1), seizure plus ketogenic diet (EXP2), the control group without ketogenic diet (CONT1), and the control plus ketogenic diet (CONT2). Neurological behavioral parameters of brain damage (plane righting reflex, cliff avoidance reflex, and open field test) were observed from P35 to P49. Morris water maze test was performed during P51-P57. Then hippocampal mossy fiber sprouting and the protein levels of ZnT3, MT3, ApoE, CLU, and ACAT-1 were detected by Timm staining and Western blot analysis, respectively. Flurothyl-induced neurobehavioral toxicology and aberrant mossy fiber sprouting were blocked by KD. In parallel with these behavioral changes, rats treated with KD (EXP2) showed a significant down-regulated expression of ZnT-3, MT-3, ApoE, clusterin, and ACAT-1 in hippocampus when compared with the non-KD-treated EXP1 group. Our findings provide support for zinc/lipid transporter signals being potential targets for the treatment of neonatal seizure-induced brain damage by KD.

Abstract  A major question concerning the learning and memory deficits characteristic of epilepsy is the relative importance of the initial insult that leads to recurrent, unprovoked seizures versus the seizures themselves. A related issue is whether seizure-induced cognitive decline is permanent or reversible when convulsions cease. To address these problems, adult rats were extensively trained in the "spatial accuracy task," a dry-land analog of the Morris water maze. This task allows the rat's estimate of the location of a hidden goal zone to be repeatedly measured within each behavioral session. One aim was to measure, in well-trained animals, the time course of any cognitive impairment caused by a daily flurothyl-induced generalized seizure over 11 days. A second aim was to look for possible recovery during 9 subsequent days with no seizures. We saw a cumulative degradation in spatial performance during the seizure days and reversal of the deficit after seizures were stopped such that performance returned to baseline. Interestingly, the rate of learning to an asymptote, the rate of performance decline during one-per-day seizures and the rate of relearning during the recovery period were all similar. Given that the hippocampus plays an important role in spatial memory and that it is the brain structure most vulnerable to abnormal excitation the implication is that the hippocampus remains essential for precise spatial navigation even after prolonged training in locating a fixed goal zone. Clinically, this finding questions the assumption that patients who experience seizures should return to a baseline cognitive level within hours.

Abstract  Hypoxic-ischemic encephalopathy is a common neonatal brain injury associated with significant morbidity and mortality despite the administration of therapeutic hypothermia (TH). Neonatal seizures and subsequent chronic epilepsy are frequent in this patient population and current treatments are partially effective. We used a neonatal murine hypoxia-ischemia (HI) model to test whether the severity of hippocampal and cortical injury predicts seizure susceptibility 8 days after HI and whether TH mitigates this susceptibility. HI at postnatal day 10 (P10) caused hippocampal injury not mitigated by TH in male or female pups. TH did not confer protection against flurothyl seizure susceptibility at P18 in this model. Hippocampal (R2 = 0.33, p = 0.001) and cortical (R2 = 0.33, p = 0.003) injury directly correlated with seizure susceptibility in male but not female pups. Thus, there are sex-specific consequences of neonatal HI on flurothyl seizure susceptibility in a murine neonatal HI model. Further studies are necessary to elucidate the underlying mechanisms of sex dimorphism in seizure susceptibility after neonatal HI.

Abstract  Zinc transporters, plasticity-related genes, and autophagic/apoptotic pathway both are associated with developmental seizure-induced brain excitotoxicity. Here, for the first time, we report the timing of expression pattern of zinc transporter 4 (ZnT-4), plasticity-related gene 3 (PRG-3), specific marker of autophagic vacuoles (LC3), and apoptotic marker caspase-3 in cerebral cortex following neonatal seizures. A seizure was induced by inhalant flurothyl daily in neonatal Sprague-Dawley rats from postnatal day 6 (P6). Rats were assigned into the recurrent-seizure group (RS, seizures induced in six consecutive days) and the control group. At 1.5 h, 3 h, 6 h, 12 h, 24 h, 48 h, 7 days, and 14 days after the last seizure, the mRNA level of the four genes in cerebral cortex was detected using RT-PCR method. At an early period 6 h or 12 h after the last seizures, both ZnT-4 and LC3 showed significantly up-regulated mRNA level while PRG-3 showed significantly down-regulated mRNA level at 12 h in cerebral cortex of RS group than those at the corresponding time point in control group. In the long-term time point of 7 days after the last seizure, the mRNA level of caspase-3 down-regulated; meanwhile, there was up-regulated mRNA level of LC-3 in RS group when compared to the control rats. This is the first report investigating the gene expression pattern of ZnT-4, PRG-3, LC-3, and caspase-3 in the developing brain. The results suggest that the disturbed expression pattern of the four genes might play a role in the pathophysiology of recurrent neonatal seizure-induced acute and long-term brain damage.