Abstract  Epilepsy in children is associated with a broad spectrum of cognitive deficits, which is associated with hippocampal mossy fiber sprouting. The underlying molecular mechanisms involved in mossy fiber sprouting in hippocampus following developmental seizures are not completely known. We studied the timing of cognitive dysfunction following neonatal seizures and the relation of this cognitive impairment to zinc transporter 1 (ZnT-1), 3 (ZnT-3), calcium/calmodulin-dependent protein kinase II (CaMK II), plasticity-related gene 1 (PRG-1) expression in hippocampus. A seizure was induced by inhalant flurothyl daily in neonatal Sprague-Dawley rats from postnatal day 6 (P6). Rats were assigned into the single-seizure group (SS), the recurrent-seizure group (RS, seizures induced in six consecutive days), and the control group. During P41-P46 and P85-P90, the rats were tested for spatial learning and memory abilities with automatic Morris water maze task. At P90, mossy fiber sprouting and gene expression in hippocampus were determined subsequently by Timm staining and RT-PCR methods. The escape latencies from the water maze were significantly longer in rats of RS group than those of the control and SS groups at d4 of the first maze test and at d3, d4 of the second maze test. As far as Spatial Probe Test was concerned, the frequency of passing through the platform quadrant was significantly decreased in RS group than that in control and SS groups in the entire two probe tests. In rats with recurrent seizures (RS group), there was an increased distribution of Timm granules in both the supragranular region of the dentate gyrus and the stratum pyramidale of CA3 subfield in RS group, while remaining barely visible in control and SS groups; the Timm scores in CA3 and dentate gyrus in the RS animals were significantly higher than that in the control and SS groups. RT-PCR densitometry analysis showed that the ratios of hippocampal ZnT-1 to beta-actin of SS and RS group were decreased significantly compared with that of control group. Meanwhile, CaMK II to beta-actin of RS group was markedly lower compared with those of SS and control groups. Our results suggest that the long-term adverse effects of recurrent neonatal seizures on cognition and mossy fiber sprouting may be associated with the down-regulated expression of ZnT-1 and CaMK II in hippocampus.

Abstract  PURPOSE:   Rapamycin (RAP) has certain antiepileptogenic features. However, it is unclear whether these effects can be explained by the anticonvulsant action of RAP, which has not been studied. To address this question, we tested potential anticonvulsant effects of RAP in immature and adult rats using different seizure models and treatment paradigms. In addition, we studied changes in the expression of neuropeptide Y (NPY) induced by RAP, which may serve as an indirect target of the RAP action.

METHODS:   A complex approach was adopted to evaluate the anticonvulsant potential of RAP: We used flurothyl-, pentylenetetrazole (PTZ)-, N-methyl-D-aspartate (NMDA)-, and kainic acid (KA)-induced seizures to test the effects of RAP using different pretreatment protocols in immature and adult rats. We also evaluated expression of NPY within the primary motor cortex, hippocampal CA1, and dentate gyrus (DG) after different pretreatments with RAP in immature rats.

KEY FINDINGS:   We found the following: (1) RAP administered with short-term pretreatment paradigms has a weak anticonvulsant potential in the seizure models with compromised inhibition. (2) Lack of RAP efficacy correlates with decreased NPY expression in the cortex, CA1, and DG. Specifically in immature rats, a single dose of RAP (3 mg/kg) 4 or 24 h before seizure testing had anticonvulsant effects against PTZ-induced seizures. In the flurothyl seizure model only the 4-h pretreatment with RAP was anticonvulsant in the both age groups. Short-term pretreatments with RAP had no effects against NMDA- and KA-induced seizures tested in immature rats. Long-term pretreatments with RAP over 8 days did not show beneficial effect in all tested seizure models in developing rats. Moreover, the long-term pretreatment with RAP had a slight proconvulsant effect on KA-induced seizures. In immature rats, any lack of anticonvulsant effect (including proconvulsant effect of multiple doses of RAP) was associated with downregulation of NPY expression in the cortex and DG. In immature animals, after a single dose of RAP with 24 h delay, we found a decrease of NPY expression in DG, and CA1 as well.

SIGNIFICANCE:   Our data show weak age-, treatment paradigm-, and model-specific anticonvulsant effects of RAP as well as loss of those effects after long-term RAP pretreatment associated with downregulation of NPY expression. These findings suggest that RAP is a poor anticonvulsant and may have beneficial effects only against epileptogenesis. In addition, our data present new insights into mechanisms of RAP action on seizures indicating a possible connection between mammalian target of rapamycin (mTOR) signaling and NPY system.

Abstract  Synaptically released zinc has neuromodulatory capabilities that could result in either inhibition or enhancement of neuronal excitability. To determine the net effects of vesicular zinc release in the brain in vivo, we examined seizure susceptibility and seizure-related neuronal damage in mice with targeted disruption of the gene encoding the zinc transporter, ZnT3 (ZnT3-/- mice). ZnT3-/- mice, which lack histochemically reactive zinc in synaptic vesicles, had slightly higher thresholds to seizures elicited by the GABA(A) antagonist, bicuculline, and no differences in seizure threshold were seen in response to pentylenetetrazol or flurothyl. However, ZnT3-/- mice were much more susceptible than wild-type mice to limbic seizures elicited by kainic acid, suggesting that the net effect of hippocampal zinc on acute seizures in vivo is inhibitory. The hippocampi of ZnT3-/- mice showed typical seizure-related neuronal damage in response to kainic acid, demonstrating that damage to the targets of zinc-containing neurons can occur independently of synaptically released zinc. Mice lacking the neuronal zinc-binding protein metallothionein III (MT-III) are also more susceptible to kainic acid-induced seizures. Double knockout (ZnT3 and MT3) mice show the same response to kainic acid as ZnT3-/- mice, suggesting that ZnT3 and MT-III function in the same pathway.

Abstract  GABAergic transmission in the substantia nigra pars reticulata (SNR) has an important role in the control of experimental seizures. In the flurothyl seizure model, SNR microinjection of the selective GABAA receptor agonist muscimol results in a biphasic dose-response curve in adults: Intermediate doses are anticonvulsant, but high doses have proconvulsant effects. Another GABAA agonist, THIP (4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol), also produces anticonvulsant effects at lower doses, whereas higher doses tend to produce a proconvulsant effect. In 16-day-old rat pups, no anticonvulsant but only proconvulsant effects of muscimol occur, and at lower doses than in adults. These data suggest that the immature SNR is significantly more sensitive to the proconvulsant effects of GABAA receptor agonists than is the SNR of adults. We hypothesize that the age-related differences in nigral GABAergic response may be due to ontogenic changes in GABAA-sensitive neuronal circuits in the SNR.

Abstract  Prolonged seizures in immature rats result in minimal behavioral consequences when the animals are studied later in life. Likewise, early-onset seizures are associated with minimal morphological changes. However, it is known that seizures early in life result in changes in the brain that make it more vulnerable to subsequent seizure-induced injury (the so-called two-hit hypothesis). Whether this heightened vulnerability occurs immediately after the first seizure is not known. In this study, immature rats were exposed to status epilepticus (SE) followed by a series of 25 flurothyl-induced seizures, SE alone, 25 flurothyl-induced seizures alone, or no seizures. Rats exposed to SE and flurothyl seizures performed significantly poorer in the water maze 2 weeks following the last seizure compared with the other groups. No histological lesions were seen in any of the four groups. This study suggests that SE renders the immature brain vulnerable to further seizure-induced injury and this enhanced vulnerability occurs very quickly after the SE.

Abstract  A model for chronic treatment of rats with sodium valproate has been developed balancing efficacy, toxicity and dose control. The dose (300 mg/kg) and frequency (every 8 h) selected were somewhat toxic as measured by weight gain and failed to provide continuous protection against Indoklon induced seizures but yielded plasma valproate levels near the range of therapeutic human levels. Chronic treatment of rats at this dose and frequency yielded a significant negative correlation between weight gain and length of treatment as well as a significant negative correlation between plasma valproate concentration and length of treatment. It was concluded that due to the short half-life of valproate in rats it is impossible to maintain continuously protective, nontoxic levels of valproate with a reasonable frequency (every 8 h) of controlled dose administration.

Abstract  Potassium channels play a critical role in limiting neuronal excitability. Mutations in certain voltage-gated potassium channels have been associated with hyperexcitable phenotypes in both humans and animals. However, only recently have mutations in potassium channel genes (i.e. KCNQ2 and KCNQ3) been discovered in a human epilepsy, benign familial neonatal convulsions. Recently, it has been reported that mice lacking the voltage-gated Shaker-like potassium channel Kv1.1 alpha-subunit develop recurrent spontaneous seizures early in postnatal development. The clinical relevance of the Kv1.1 knockout mouse has been underscored by a recent report of epilepsy occurring in a family affected by mutations in the KCNA1 locus (the human homologue of Kv1.1) which typically cause episodic ataxia and myokymia. Here we summarize preliminary studies characterizing the developmental changes in seizure susceptibility and neuronal activation in the three genotypes of Kv1.1 mice (-/-, +/-, +/+). Using behavioral and immediate-early gene indicators of regional brain excitability, we have found that a seizure-sensitive predisposition exists in Kv1.1 -/- animals at a very young age (P10), before either spontaneous seizure activity or changes in c-fos mRNA expression can be demonstrated. Kv1.1 +/- mice, although behaviorally indistinguishable from wild types, also have an increased susceptibility to seizures at a similar early age. The Kv1. 1 knockout mouse possesses many features desirable in a developmental animal epilepsy model and represents a clinically relevant model of early-onset epilepsies.

Abstract  Ketogenic diet (KD) is a high fat, low carbohydrate diet used to treat children with epilepsy that are refractory to conventional antiepileptic drugs (AEDs). The anticonvulsant mechanism of the KD is unknown. To determine if the noradrenergic system has a role in mediating the anticonvulsant action of the KD, dopamine beta-hydroxylase knockout (Dbh -/-) mice that lack norepinephrine (NE) and Dbh +/- littermates that have normal NE content were fed either a standard rodent chow or the KD. When exposed to the convulsant flurothyl, Dbh +/- mice fed the KD had significantly longer latencies to myoclonic jerk (MJ) and generalized clonic-tonic (CT) seizures than Dbh +/- mice fed normal chow. In contrast, Dbh -/- mice fed the KD had seizure latencies to both MJ and CT comparable to Dbh -/- mice fed normal chow. These results suggest that an intact, functional noradrenergic nervous system is required for the KD to exert an anticonvulsant effect.

Abstract  The anticonvulsant and neuroprotective activity of dextromethorphan (DM, [+]-3-methyl-17-methylmorphinan) may be, in part, due to its metabolism to the phencyclidine hydrochloride-like compound dextrorphan (DX). We evaluated the anticonvulsant activity and neurological impairing effects in rats of three novel analogs of DM which, based upon their position-3 substituents, would either not be expected to be metabolized to DX or might do so at a reduced rate. The DM analogs were determined to be more potent and more efficacious than DM against maximal electroshock convulsions; two of the analogs, namely [(+)-3-ethoxy-17-methylmorphinan] and [(+)-3-(2-propoxy)-17-methyl-morphinan], were equipotent to DX. [(+)-3-(Ethoxy-17-methylmorphinan], and [(+)-3-(2-propoxy)-17-methylmorphinan) exhibited a duration of action (1-2 hr) slightly longer than DX (0.5-1 hr) and similar to DM (2-4 hr). The anticonvulsant effect of [(+)-3-amino-17-methylmorphinan] persisted 4-6 hr. Against flurothyl convulsions DM was proconvulsant, DX was anticonvulsant and the DM analogs were inactive. In contrast, N-methyl-D-aspartate convulsions were antagonized by i.c.v. pretreatment with DM and the DM analogs, albeit with a potency approximately 10 times less than that of DX. Results of rotarod performance testing further distinguished the analogs from DM, DX or the anticonvulsant drug diazepam. No behavioral impairment was observed at the highest doses tested of each of the DM analogs, resulting in protective indices (i.e., rotarod TD50/maximal electroshock anticonvulsant ED50) greatly exceeding DM, DX or clinical anticonvulsant drugs. The results of this study establish these 3-substituted DM analogs as novel anticonvulsants exhibiting improved potency, efficacy, duration and side-effect profiles.

Abstract  The present investigation was designed to study the effect of chemically induced seizures on cerebral hypoxic-ischemic (HI) damage in immature animals. Accordingly, cerebral HI was produced in 7-day postnatal (p7) rats and p13 rats by combined unilateral common carotid artery ligation and hypoxia with 8% oxygen. Seizures were induced chemically by the subcutaneous injection of kainic acid (KA) or inhalation of flurothyl vapor. Three types of experiments were conducted in each age group and for each convulsant. In some animals (group 1), seizures were produced at 24 h and again at 6 h prior to HI. In groups 2 and 3, seizures were induced 2 h or 24 h post HI, respectively. The results indicate that in group 1 animals, the first seizure significantly reduced duration of the second seizure challenge 18 h later at both p7 and p13 (p=0.001). Histologic examination of brains of animals in group 1 subjected to seizures prior to HI and their HI-only controls showed that seizures prior to HI conferred protection against cerebral damage. This effect was significant for flurothyl seizures in p13 rats for all cerebral regions, especially hippocampal CA1 (p=0.0004), and in p7 rats for hippocampus (p=0.04) and particularly cerebral cortex (p=0.007). For KA seizures, the protective effect was only significant in p13 rats and was limited to hippocampal CA regions and subiculum (p=0.0009). Histologic assessment of cerebral lesions of p7 and p13 rats in the other two groups showed no significant difference between the animals subjected to seizures 2 h or 24 h post HI and their HI-only controls (p>0.05). In conclusion, the results of the present study provide no evidence that seizures in early postnatal development aggravate pre-existing cerebral HI damage. They do suggest that seizures prior to HI or prior to a second seizure confer tolerance to both conditions.

Abstract  Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by multi-organ pathologies. Most TSC patients exhibit seizures, usually starting in early childhood. The neuropathological hallmarks of the disease - cortical tubers, containing cytopathological neuronal and glial cell types - appear to be the source of seizure initiation. However, the contribution of these aberrant cell populations to TSC-associated epilepsies is not fully understood. To gain further insight, investigators have attempted to generate animal models with TSC-like brain abnormalities. In the current study, we focused on the Eker rat, in which there is a spontaneous mutation of the TSC2 gene (TSC2+/-). We attempted to exacerbate TSC-like brain pathologies with a "second-hit" strategy - exposing young pups to ionizing irradiation of different intensities, and at different developmental timepoints (between E18 and P6). We found that the frequency of occurrence of dysmorphic neurons and giant astrocytes was strongly dependent on irradiation dose, and weakly dependent on timing of irradiation in Eker rats, but not in irradiated normal controls. The frequency of TSC-like pathology was progressive; there were many more abnormal cells at 3 months compared to 1 month post-irradiation. Measures of seizure propensity (flurothyl seizure latency) and brain excitability (paired-pulse and post-tetanic stimulation studies in vitro), however, showed no functional changes associated with the appearance of TSC-like cellular abnormalities in irradiated Eker rats.

Abstract  OBJECTIVE: To determine changes in glucose metabolism and the enzymes involved in the hippocampus ictally and postictally in the acute mouse flurothyl seizure model.

METHODS: [U-13 C]-Glucose was injected (i.p.) prior to, or following a 5 min flurothyl-induced seizure. Fifteen minutes later, mice were killed and the total metabolite levels and % 13 C enrichment were analyzed in the hippocampal formation using gas chromatography-mass spectrometry. Activities of key metabolic and antioxidant enzymes and the phosphorylation status of pyruvate dehydrogenase were measured, along with lipid peroxidation.

RESULTS: During seizures, total lactate levels increased 1.7-fold; however, [M + 3] enrichment of both lactate and alanine were reduced by 30% and 43%, respectively, along with a 28% decrease in phosphofructokinase activity. Postictally the % 13 C enrichments of all measured tricarboxylic acid (TCA) cycle intermediates and the amino acids were reduced by 46-93%. At this time, pyruvate dehydrogenase (PDH) activity was 56% of that measured in controls, and there was a 1.9-fold increase in the phosphorylation of PDH at ser232. Phosphorylation of PDH is known to decrease its activity.

SIGNIFICANCE: Here, we show that the increase of lactate levels during flurothyl seizures is from a source other than [U-13 C]-glucose, such as glycogen. Surprisingly, although we saw a reduction in phosphofructokinase activity during the seizure, metabolism of [U-13 C]-glucose into the TCA cycle seemed unaffected. Similar to our recent findings in the chronic phase of the pilocarpine model, postictally the metabolism of glucose by glycolysis and the TCA cycle was impaired along with reduced PDH activity. Although this decrease in activity may be a protective mechanism to reduce oxidative stress, which is observed in the flurothyl model, ATP is critical to the recovery of ion and neurotransmitter balance and return to normal brain function. Thus we identified promising novel strategies to enhance energy metabolism and recovery from seizures.

Abstract  The effects of valproate (VPA), zonisamide (ZNS), and phenytoin (PHT) on flurothyl (FE)-induced generalized seizure were investigated in mice. In the FE kindling model, eight daily FE-induced generalized clonic seizures followed by a 28-day stimulation-free interval converted the type of seizure expressed in response to FE from clonic to tonic. In an acute FE trial experiment, the latencies of clonic and tonic seizures were prolonged significantly and dose-dependently by the administration of VPA. ZNS and PHT did not show any effect on the latencies of tonic seizure. When the same three drugs were administered to mice daily for 8 days prior to the FE trial, changes in the seizure phenotypes from clonic to tonic seizure were significantly inhibited by VPA and ZNS, but not by PHT. These results suggest that VPA and ZNS possess significant antiepileptogenic properties. PHT apparently does not share this property to the same degree in the present FE-induced model.

Abstract  PURPOSE: Status epilepticus (SE) commonly occurs in children, whereas ischemic stroke is the most frequent neurologic insult in adults. The purpose of this study was to determine the effect of SE induced in immature (15 days old; PN15) male rats, on susceptibility to subsequent transient focal cerebral ischemia induced in adulthood.

METHODS: SE was induced by flurothyl ether (FE) or kainic acid (KA). Rats that did not develop seizures after FE or KA served as controls. Five weeks later, the now-adult rats were subjected to middle cerebral artery occlusion (MCAo) for 1 or 2 h by using the intraluminal filament technique. The extent of the infarct volume was evaluated 24 h later.

RESULTS: In rats submitted to 1-h-long FE-SE, the volume of infarction was significantly reduced compared with that in rats exposed to FE without SE. Longer duration of FE-SE was acutely lethal. KA-SE induced prolonged behavioral SE (156 +/- 17.5 min). In these rats, the volume of infarction was significantly larger compared with that in rats that did not show any electrographic seizures after KA administration. Comparison of FE and KA groups revealed that differences in the size of infarction were confined into cortical areas served by the MCA. Neither type of SE induced any obvious histologic changes in these neocortical regions before stroke induction.

CONCLUSIONS: Early in life, SE can influence the outcome of a subsequent focal ischemic insult in adulthood. The extent of the infarct is related to the duration and cause of SE. Prolonged SE induced by KA worsens the outcome, whereas FE-SE has a neuroprotective effect.

Abstract  There is now considerable evidence that the N-methyl-D-aspartic acid receptor is important in the genesis of seizures. One of the selective antagonist of the NMDA receptor is 2-amino-7-phosphonoheptanoic acid (APH). In this study we evaluated the effects of intracerebroventricular (i.c.v.) administration of APH on seizure susceptibility in both prepubescent and mature rats using the rapid kindling and flurothyl ether seizure models. Both the immature and mature animals receiving APH kindled at a significantly slower rate than control animals receiving phosphate-buffered saline. APH also demonstrated a significant anticonvulsant effect against flurothyl-induced seizures in both the immature and mature animals. This study supports prior work that selective NMDA receptor antagonists such as APH may have promise as potential antiepileptic agents.

Abstract  OBJECTIVE: To investigate the effets of flurothyl-induced neonatal recurrent seizures on glucocorticoid receptor (GR) expression in the rat brain.

METHODS: Forty-eight seven-day-old Sprague-Dawley rats were randomly divided into two groups: control and seizure. Seizures were induced by inhalant flurothyl daily for six consecutive days. Brains were sampled on postnatal days 13, 15 and 19. The expression of GR protein in the cerebral cortex was detected by Western blot and immunohistochemical method.

RESULTS: The expression of GR in the cerebral cortical plasma protein was significantly lower in the seizure group than in the control group on postnatal day 15. The expression of GR protein in the cerebral cortical nuclear protein decreased significantly in the seizure group compared with that in the control group on postnatal days 15 and 19 (p<0.05). Compared to the control group, the accumulated optical density (AOD) of GR immunoreactivity (IR) decreased significantly in the parietal cortex on postnatal day 13 (p<0.05), the AOD of GR IR decreased significantly in the parietal cortex and the temporal cortex on postnatal day 15 (p<0.05), and the AOD of GR IR decreased significantly in the parietal cortex, temporal cortex and the frontal cortex in the seizure group on postnatal day 19 (p<0.05).

CONCLUSIONS: Recurrent seizures in neonatal rats result in abnormal GR expression in the cerebral cortex which might play an important role in short-term brain injury induced by early recurrent seizures.

Abstract  The effect of pregnancy on chemically-induced seizures in mice was studied. Latencies to myoclonic and clonic seizures induced by inhalation of flurothyl were significantly reduced at days 12 through 18 of gestation. Parturition resulted in a return of seizure susceptibility to control levels. The possibility that this effect might be mediated by decreased neurotransmitter levels subsequent to the decreased vitamin B6 levels which are known to occur during pregnancy was suggested. A pregnancy-associated liver cytosolic aldehyde dehydrogenase (pi-AlDH) utilized pyridoxal as a substrate, and the peak of pi-AlDH activity was shown to coincide with the peak of seizure susceptibility. The activity of aldehyde oxidase, the major enzyme normally responsible for the metabolism of pyridoxal, was reduced in pregnant animals. The pyridoxal 5'-phosphate synthesizing enzymes, pyridoxal kinase and pyridoxamine phosphate oxidase, were marginally increased in activity during pregnancy. It was suggested that the increased activity of pi-AlDH was indirectly responsible for the increased seizure susceptibility due to increased metabolism of pyridoxal.

Abstract  It has been hypothesized that the Long-Sleep and Short-Sleep mouse lines were bidirectionally selected for high and low brain excitability, and further, that these differences are mediated by the benzodiazepine/gamma-aminobutyric acid (GABA) receptor-chloride channel complex. Hence, mice from both lines were administered seven convulsants (bicuculline, pentylenetetrazol, 3-carbomethoxy-beta-carboline, picrotoxin, caffeine, flurothyl and strychnine) and myoclonic and clonic seizure latencies recorded. Supporting the original hypothesis, the results show that the two lines were differentiated by all of the convulsants and that in response to the drugs, three distinct convulsive patterns were found. Nevertheless, a simple genetic model accounting for these results was not evident. To further clarify these susceptibility patterns, a convulsant representing each of these patterns (bicuculline, pentylenetetrazol or caffeine) was administered in conjunction with the anticonvulsant-barbiturate phenobarbital or the benzodiazepine antagonist Ro 15-1788. Irrespective of the convulsant given, phenobarbital attenuated both myoclonus and clonus subsequent to all convulsants, while Ro 15-1788 had a more discrete anticonvulsant profile.

Abstract  Young chicks survive flurothyl vapor concentrations that are lethal to mice. For a 10-min exposure, the LD50 concentrations (% v/v) of flurothyl are 10.1% in chicks and 0.33% in mice; the convulsant concentrations (CD50) are 0.49% in chicks and 0.32% in mice. The remarkable resistance of young chicks to flurothyl toxicity may be related to their known resistance to hypoxic death.