Timing of cognitive deficits following neonatal seizures: relationship to histological changes in the hippocampus.

Neonatal seizures are frequently associated with cognitive impairment and reduced seizure threshold. Previous studies in our laboratory have demonstrated that rats with recurrent neonatal seizures have impaired learning, lower seizure thresholds, and sprouting of mossy fibers in CA3 and the supragranular region of the dentate gyrus in the hippocampus when studied as adults. The goal of this study was to determine the age of onset of cognitive dysfunction and alterations in seizure susceptibility in rats subjected to recurrent neonatal seizures and the relation of this cognitive impairment to mossy fiber sprouting and expression of glutamate receptors. Starting at postnatal day (P) 0, rats were exposed to 45 flurothyl-induced seizures over a 9-day period of time. Visual-spatial learning in the water maze and seizure susceptibility were assessed in subsets of the rats at P20 or P35. Brains were evaluated for cell loss, mossy fiber distribution, and AMPA (GluR1) and NMDA (NMDAR1) subreceptor expression at these same time points. Rats with neonatal seizures showed significant impairment in the performance of the water maze and increased seizure susceptibility at both P20 and P35. Sprouting of mossy fibers into the CA3 and supragranular region of the dentate gyrus was seen at both P20 and P35. GluR1 expression was increased in CA3 at P20 and NMDAR1 was increased in expression in CA3 and the supragranular region of the dentate gyrus at P35. Our findings indicate that altered seizure susceptibility and cognitive impairment occurs prior to weaning following a series of neonatal seizures. Furthermore, these alterations in cognition and seizure susceptibility are paralleled by sprouting of mossy fibers and increased expression of glutamate receptors. To be effective, our results suggest that strategies to alter the adverse outcome following neonatal seizures will have to be initiated during, or shortly following, the seizures.     

Long-term effects of neonatal seizures: a behavioral, electrophysiological, and histological study

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.     

Consequences of neonatal seizures in the rat: Morphological and behavioral effects

Whereas neonatal seizures are a predictor of adverse neurological outcome, there is controversy regarding whether seizures simply reflect an underlying brain injury or can cause damage. We subjected neonatal rats to a series of 25 brief flurothyl-induced seizures. Once mature the rats were compared with control littermates for spatial learning and activity level. Short-term effects of recurrent seizures on hippocampal excitation were assessed by using the intact hippocampus formal preparation and long-term effects by assessing seizure threshold. Brains were analysed for neuronal loss, sprouting of granule cell axons (mossy fibers), and neurogenesis. Compared with controls, rats subjected to neonatal seizures had impaired learning and decreased activity levels. There were no differences in paired-pulse excitation or inhibition or duration of afterdischarges in the intact hippocampal preparation. However, when studied as adults, rats with recurrent flurothyl seizures had a significantly lower seizure threshold to pentylenetrazol than controls. Rats with recurrent seizures had greater numbers of dentate granule cells and more newly formed granule cells than the controls. Rats with recurrent seizures also had sprouting of mossy fibers in CA3 and the supragranular region. Recurrent brief seizures during the neonatal period have long-term detrimental effects on behavior, seizure susceptibility, and brain development.     

N-cadherin在颞叶癫痫海马苔藓纤维出芽和突触重组中的作用

目的 探讨神经性钙粘附分子(N-cadherin)在癫痫状态后海马苔藓纤维出芽和突触重组中的作用。方法取锂一匹罗卡品诱导大鼠癫痫持续状态及慢性自发性颞叶癫痫发作期的大鼠脑片，用Timm染色和免疫组化的方法分别检测苔藓纤维出芽和N-cadherin在大鼠海马组织中的表达。结果癫痫状态后第2周和第4周的实验组大鼠可见到苔藓纤维出芽，穿越齿状回颗粒细胞层到达内分子层，并在此形成一条致密的层状带(Timm染色)。免疫组化染色发现实验组大鼠在第2周和第4周，海马齿状回内分子层可以看到强染色，并形成一条致密带，与Timm染色时观察到的条带一致。结论癫痫状态后在海马齿状回内分子层N-cadherin的表达上调．N-cadherin可能参与了癫痫后苔藓纤维出芽和突触重组过程。     

边缘癫痫实验模型海马内突触体素表达

目的探讨癫痫时突触体素(P^38)在海马表达的时间变化及意义。方法建立匹罗卡品边缘癫痫模型，用图像分析系统测定海马不同时间点P^38免疫反应吸光度值。结果P^38免疫反应性在海马呈现两次高峰：致痫后3～6h在海马门区及CA3区P^38短期升高，30～60dCA3区呈现第2次高峰。在内分子层，从第7天开始直至第60天P^38呈进行性增多，且与Neo—Timm染色结果相平行。结论P^38在海马第2次表达增高，平行于苔藓纤维出芽，与自发性发作形成有关。急性期表达增高则与癫痫持续状态的产生与维持有关。     

卡马西平对成年癫大鼠海马齿状回BrdU/NeuN表达水平及其对空间记忆的影响

目的研究卡马西平对成年癫大鼠海马齿状回新生神经元的影响及其与空间记忆之间的关系。方法采用氯化锂和匹罗卡品联合诱导大鼠癫模型,利用5-溴脱氧尿苷嘧啶与神经元核性蛋白双标记观察海马齿状回内源性神经前体细胞分化为成熟神经元的情况;利用行为学分析评价大鼠的空间记忆。结果 (1)卡马西平可增加癫大鼠海马齿状回新生成熟神经元的数量(P<0.05);(2)卡马西平对癫大鼠的空间记忆有明显改善作用(P<0.01)。结论卡马西平增加癫大鼠海马齿状回新生成熟神经元形成,是其改善癫大鼠空间记忆的可能机制之一。     

Circuit Mechanisms of Seizures in the Pilocarpine Model of Chronic Epilepsy: Cell Loss and Mossy Fiber Sprouting

We used the pilocarpine model of chronic spontaneous recurrent seizures to evaluate the time course of supragranular dentate sprouting and to assess the relation between several changes that occur in epilep tic tissue with different behavioral manifestations of this experimental model of temporal lobe epilepsy. Pilo carpine-induced status epilepticus (SE) invariably led to cell loss in the hilus of the dentate gyrus (DG) and to spontaneous recurrent seizures. Cell loss was often also noted in the DG and in hippocampal subfields CA1 and CA3. The seizures began to appear at a mean of 15 days after SE induction (silent period), recurred at variable frequencies for each animal, and lasted for as long as the animals were allowed to survive (325 days). The granule cell layer of the DG was dispersed in epileptic animals, and neo-Timm stains showed supra-and intragranular mossy fiber sprouting. Supragranular mossy fiber sprout ing and dentate granule cell dispersion began to appear early after SE (as early as 4 and 9 days, respectively) and reached a plateau by 100 days. Animals with a greater degree of cell loss in hippocampal field CAS showed later onset of chronic epilepsy (r= 0.83, p < 0.0005), suggest ing that CA3 represents one of the routes for seizure spread. These results demonstrate that the pilocarpine model of chronic seizures replicates several of the fea tures of human temporal lobe epilepsy (hippocampal cell loss, suprar and intragranular mossy fiber sprouting, den tate granule cell dispersion, spontaneous recurrent sei zures) and that it may be a useful model for studying this human condition. The results also suggest that even though a certain amount of cell loss in specific areas may be essential for chronic seizures to occur, excessive cell loss may hinder epileptogenesis.     

Localized overexpression of FGF-2 and BDNF in hippocampus reduces mossy fiber sprouting and spontaneous seizures up to 4 weeks after pilocarpine-induced status epilepticus

Purpose: We have recently reported that viral vector–mediated supplementation of fibroblast growth factor‐2 (FGF‐2) and brain‐derived neurotrophic factor (BDNF) in a lesioned, epileptogenic rat hippocampus limits neuronal damage, favors neurogenesis, and reduces spontaneous recurrent seizures. To test if this treatment can also prevent hippocampal circuit reorganization, we examined here its effect on mossy fiber sprouting, the best studied form of axonal plasticity in epilepsy. Methods: A herpes‐based vector expressing FGF‐2 and BDNF was injected into the rat hippocampus 3 days after an epileptogenic insult (pilocarpine‐induced status epilepticus). Continuous video–electroencephalography (EEG) monitoring was initiated 7 days after status epilepticus, and animals were sacrificed at 28 days for analysis of cell loss (measured using NeuN immunofluorescence) and mossy fiber sprouting (measured using dynorphin A immunohistochemistry). Key Findings: The vector expressing FGF‐2 and BDNF decreased both mossy fiber sprouting and the frequency and severity of spontaneous seizures. The effect on sprouting correlated strictly with the cell loss in the terminal fields of physiologic mossy fiber innervation (mossy cells in the dentate gyrus hilus and CA3 pyramidal neurons). Significance: These data suggest that the supplementation of FGF‐2 and BDNF in an epileptogenic hippocampus may prevent epileptogenesis by decreasing neuronal loss and mossy fiber sprouting, that is, reducing some forms of circuit reorganization.     

新生期大鼠反复痫性发作的形态学及行为学结果与糖皮质激素水平升高有关

目的探讨新生期大鼠反复痫性发作后的形态学,行为学以及糖皮质激素水平的变化。方法64只出生后一天的Wistar大鼠随机分为惊厥组40只和对照组24只。惊厥组的新生鼠在出生后1天（P1）、4天（P4）、7天（P7）给予腹腔注射匹罗卡品,制备新生鼠癫痫模型;对照组的新生鼠腹腔注射生理盐水。惊厥组分别在第3次致痫后在即刻（Ⅰ组）、第4天（Ⅱ组）、第14天（Ⅲ组）、第42天（Ⅳ组）四个时间点处死,各时间点设相应对照组,处死前36h惊厥组和对照组的大鼠腹腔注射BrdU。所有大鼠处死前均取血检测糖皮质激素。第Ⅳ组从P40开始进行Morris水迷宫试验。结果新生鼠3次发作后即刻和第4天与相应日龄对照组相比,齿状回BrdU阳性细胞数明显减少（P〈0．05）,而癫痫发作后14天和42天BrdU阳性细胞数增加,但发作后14天差异无统计学意义（P〉0．05）。在4天的Morris水迷宫试验中,匹罗卡品处理组大鼠到达平台的时间均长于对照组,但是只有第1天和第2天有统计学意义（P〈0．05）。检测结果表明高水平的糖皮质激素一直持续到发作后第4天,糖皮质激素水平与BrdU阳性细胞数呈负相关。结论新生大鼠反复痫性发作会造成早期神经发生减少,而后期神经发生增加;造成大鼠成年后认知功能缺陷;造成糖皮质激素水平增高,这与痫性大鼠形态学和行为学方面的改变有关。     

Locus Coeruleus and Neuronal Plasticity in a Model of Focal Limbic Epilepsy

Summary:  Purpose: A lesion of the noradrenergic nucleus Locus Coeruleus (LC) converts sporadic seizures evoked by microinfusion of bicuculline into the anterior piriform cortex (APC) of rats into limbic status epilepticus (SE). The purpose of this study was to evaluate the chronic effects of this new model of SE on the onset of secondary epileptogenesis. We further related the loss of noradrenaline (NE) with hippocampal mossy fiber sprouting.
Methods: Male Sprague Dawley rats were treated with systemic saline or DSP-4 (a neurotoxin selective for noradrenergic terminals originating from the LC), microinfused with bicuculline into the APC three days later, and sacrificed after 45 days. Naïve and DSP-4 pretreated sham-operated rats served as respective controls. The following evaluations were performed: (a) monitoring of acute seizures and delayed occurrence of spontaneous recurrent seizures (SRS); (b) NE levels in the hippocampus, frontal and olfactory cortex; (c) occurrence of mossy fiber sprouting into the inner molecular layer of the dentate gyrus of the dorsal hippocampus.
Results: In 30% of rats lacking noradrenergic terminals, SE evoked from the APC was followed by SRS. Conversely, seizures evoked in intact rats did not result in chronic epileptogenesis. Seizures/SE did not modify NE levels as compared with baseline levels both in naïve and DSP-4-pretreated rats. Rats undergoing SE following DSP-4 + bicuculline developed SRS which were accompanied by hippocampal mossy fiber sprouting.
Conclusions: Noradrenergic loss converts focally induced sporadic seizures into an epileptogenic SE, which is accompanied by mossy fiber sprouting within the dentate gyrus.     

TsTx toxin isolated from Tityus serrulatus scorpion venom induces spontaneous recurrent seizures and mossy fiber sprouting

PURPOSE: To characterize the long-term behavioral, electroencephalographic (EEG) and histopathologic features after a single TsTx microinjection into the hippocampus of rats. METHODS: TsTx, 2 microg, or 1 microl of 0.1 M phosphate buffer was injected into the right dorsal hippocampus of the rat. EEG records and behavioral observations were made over a period of 10 h after injection. For a period of 4 months, the animals were observed for the occurrence of convulsive seizures. At the end of the experiment, the brains were processed by the neo-Timm and Nissl methods. RESULTS: After intrahippocampal TsTx injection, three distinct phases were observed: (a) an immediate period that lasted 1 day, during which the motor and electrographic seizures characteristic of status epilepticus (SE) were seen; (b) a silent period (31-49 days), characterized by normal EEG and behavior; and (c) a period of spontaneous recurrent seizures (SRSs). The seizure frequency was one to two per week. Four months after TsTx injection, hippocampal neuronal loss and mossy fiber sprouting in the supragranular layer of the dentate gyrus were observed. CONCLUSIONS: The SRSs observed in this study may be associated with the TsTx-induced SE and brain damage. All animals injected with the toxin showed massive pyramidal neuronal loss in the dorsal hippocampus as well as intense gliosis and atrophy. Mossy fiber sprouting in the supragranular layer of the dentate gyrus was observed in those animals that had SRSs. The effects observed may be due, at least in part, to TsTx-enhanced release of glutamate in hippocampal pathways.     

Is mossy fiber sprouting present at the time of the first spontaneous seizures in rat experimental temporal lobe epilepsy?

The contribution of mossy fiber sprouting to the generation of spontaneous seizures in the epileptic brain is under dispute. The present study addressed this question by examining whether sprouting of mossy fibers is present at the time of appearance of the first spontaneous seizures in rats, and whether all animals with increased sprouting have spontaneous seizures. Epileptogenesis was induced in 16 rats by electrically stimulating the lateral nucleus of the amygdala for 20-30 min until the rats developed self-sustained status epilepticus (SSSE). During and after SSSE, rats were monitored in long-term by continuous video-electroencephalography until they developed a second spontaneous seizure (8-54 days). Thereafter, monitoring was continued for 11 days to follow seizure frequency. The density of mossy fiber sprouting was analyzed from Timm-stained preparations. The density of hilar neurons was assessed from thionin-stained sections. Of 16 rats, 14 developed epilepsy. In epileptic rats, the density of mossy fiber sprouting did not correlate with the severity or duration (115-620 min) of SSSE, delay from SSSE to occurrence of first (8-51 days) or second (8-54 days) spontaneous seizure, or time from SSSE to perfusion (20-63 days). In the temporal end of the hippocampus, the sprouting correlated with the severity of neuronal damage (ipsilateral: r = -0.852, P < 0.01 contralateral: r = -0.748, P < 0.01). The two animals without spontaneous seizures also had sprouting. Increased density of sprouting in animals without seizures, and its association with the severity of neuronal loss was confirmed in another series of 30 stimulated rats that were followed-up with video-EEG monitoring for 60 d. Our data indicate that although mossy fiber sprouting is present in all animals with spontaneous seizures, its presence is not necessarily associated with the occurrence of spontaneous seizures.     

Status epilepticus-induced hilar basal dendrites on rodent granule cells contribute to recurrent excitatory circuitry

Mossy fiber sprouting into the inner molecular layer of the dentate gyrus is an important neuroplastic change found in animal models of temporal lobe epilepsy and in humans with this type of epilepsy. Recently, we reported in the perforant path stimulation model another neuroplastic change for dentate granule cells following seizures: hilar basal dendrites (HBDs). The present study determined whether status epilepticus-induced HBDs on dentate granule cells occur in the pilocarpine model of temporal lobe epilepsy and whether these dendrites are targeted by mossy fibers. Retrograde transport of biocytin following its ejection into stratum lucidum of CA3 was used to label granule cells for both light and electron microscopy. Granule cells with a heterogeneous morphology, including recurrent basal dendrites, and locations outside the granule cell layer were observed in control preparations. Preparations from both pilocarpine and kainate models of temporal lobe epilepsy also showed granule cells with HBDs. These dendrites branched and extended into the hilus of the dentate gyrus and were shown to be present on 5% of the granule cells in pilocarpine-treated rats with status epilepticus, whereas control rats had virtually none. Electron microscopy was used to determine whether HBDs were postsynaptic to axon terminals in the hilus, a site where mossy fiber collaterals are prevalent. Labeled granule cell axon terminals were found to form asymmetric synapses with labeled HBDs. Also, unlabeled, large mossy fiber boutons were presynaptic to HBDs of granule cells. These results indicate that HBDs are present in the pilocarpine model of temporal lobe epilepsy, confirm the presence of HBDs in the kainate model, and show that HBDs are postsynaptic to mossy fibers. These new mossy fiber synapses with HBDs may contribute to additional recurrent excitatory circuitry for granule cells.     

Amygdala kindling develops without mossy fiber sprouting and hippocampal neuronal degeneration in rats

Repeated electrical stimulation of limbic structures has been reported to produce the kindling effect together with morphological changes in the hippocampus such as mossy fiber sprouting and/or neuronal loss. However, to argue against a causal role of these neuropathological changes in the development of kindling-associated seizures, we examined mossy fiber sprouting in amygdala (AM)-kindled rats using Timm histochemical staining, and evaluated the hippocampal neuronal degeneration in AM-kindled rats by terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labelling (TUNEL). Amygdala kindling was established by 10.3 +/- 0.7 electrical stimulations, and no increase in Timm granules (neuronal sprouting) was observed up to the time of acquisition of a fully kindled state. However, the density and distribution of Timm granules increased significantly in the dentate gyrus compared with unkindled rats after 29 after-discharges or more than 10 kindled convulsions. In addition, no significant increase in TUNEL-positive cells was found in the hilar polymorphic neurons or in CA3 pyramidal neurons of the kindled rats that had fewer than 29 after-discharges. However, a significant increase of TUNEL-positive cells was found in the granule cell layer in the dentate gyrus of the stimulated side after 18 after-discharges or 10 kindled convulsions. Our result show that AM kindling develops without evidence of mossy fiber sprouting, and that mossy fiber sprouting may appear after repeated kindled convulsions, following death of the granule cells in the dentate gyrus.     

Synaptic connections of hilar basal dendrites of dentate granule cells in a neonatal hypoxia model of epilepsy

Numerous animal models of epileptogenesis demonstrate neuroplastic changes in the hippocampus. These changes occur not only for the mature neurons and glia, but also for the newly generated granule cells in the dentate gyrus. One of these changes, the sprouting of mossy fiber axons, is derived predominantly from newborn granule cells in adult rats with pilocarpine-induced temporal lobe epilepsy. Newborn granule cells also mainly contribute to another neuroplastic change, hilar basal dendrites (HBDs), which are synaptically targeted by mossy fibers in the hilus. Both sprouted mossy fibers and HBDs contribute to recurrent excitatory circuitry that is hypothesized to be involved in increased seizure susceptibility and the development of spontaneous recurrent seizures (SRS) that occur following the initial pilocarpine-induced status epilepticus. Considering the putative role of these neuroplastic changes in epileptogenesis, a critical question is whether similar anatomic phenomena occur after epileptogenic insults to the immature brain, where the proportion of recently born granule cells is higher due to ongoing maturation. The current study aimed to determine if such neuroplastic changes could be observed in a standardized model of neonatal seizure-inducing hypoxia that results in development of SRS. We used immunoelectron microscopy for the immature neuronal marker doublecortin to label newborn neurons and their HBDs following neonatal hypoxia. Our goal was to determine whether synapses form on HBDs from neurons born after neonatal hypoxia. Our results show a robust synapse formation on HBDs from animals that experienced neonatal hypoxia, regardless of whether the animals experienced tonic-clonic seizures during the hypoxic event. In both cases, the axon terminals that synapse onto HBDs were identified as mossy fiber terminals, based on the appearance of dense core vesicles. No such synapses were observed on HBDs from newborn granule cells obtained from sham animals analyzed at the same time points. This aberrant circuit formation may provide an anatomic substrate for increased seizure susceptibility and the development of epilepsy.     

新生期大鼠反复痫性发作的形态学及行为学结果与糖皮质激素水平升高有关(英文)

目的探讨新生期大鼠反复痫性发作后的形态学,行为学以及糖皮质激素水平的变化。方法64只出生后一天的Wistar大鼠随机分为惊厥组40只和对照组24只。惊厥组的新生鼠在出生后1天(P1)、4天(P4)、7天(P7)给予腹腔注射匹罗卡品,制备新生鼠癫痫模型;对照组的新生鼠腹腔注射生理盐水。惊厥组分别在第 3次致痫后在即刻(Ⅰ组)、第4 天(Ⅱ组)、第14 天(Ⅲ组)、第42天(Ⅳ组)四个时间点处死,各时间点设相应对照组,处死前36 h惊厥组和对照组的大鼠腹腔注射BrdU。所有大鼠处死前均取血检测糖皮质激素。第Ⅳ组从P40开始进行Morris水迷宫试验。结果新生鼠3次发作后即刻和第4天与相应日龄对照组相比,齿状回BrdU阳性细胞数明显减少(P<0.05),而癫痫发作后14天和42天BrdU阳性细胞数增加,但发作后14天差异无统计学意义(P>0.05)。在4天的Morris水迷宫试验中,匹罗卡品处理组大鼠到达平台的时间均长于对照组,但是只有第1天和第2天有统计学意义(P<0.05)。检测结果表明高水平的糖皮质激素一直持续到发作后第4天,糖皮质激素水平与BrdU阳性细胞数呈负相关。结论新生大鼠反复痫性发作会造成早期神经发生减少,而后期神经发生增加;造成大鼠成年后认知功能缺陷;造成糖皮质激素水平增高,这与痫性大鼠形态学和行为学方面的改变有关。     

Effects of herbimycin A in the pilocarpine model of temporal lobe epilepsy

Pilocarpine-induced status epilepticus (SE) causes widespread tyrosine phosphorylation in the brain. It has been postulated that this intracellular signal may mediate potentially epileptogenic changes in the morphology and physiology of particular brain regions, including the hippocampus. The present study evaluated the effects of herbimycin A, a protein tyrosine kinase (PTK) inhibitor, over the acute (during which intense biochemical and electrophysiological activation occurs) and the chronic phase (characterized by spontaneous and recurrent epileptic seizures and the presence of synaptic reorganization, e.g., mossy fiber sprouting) of the pilocarpine model of epilepsy. The administration of a single dose of 1.74 nmol of herbimycin A (i.c.v., 5 microL) 5 min after the onset of SE did not change the acute behavioral manifestation of seizures despite significantly decreasing c-Fos immunoreactivity in different areas of the hippocampus and of the limbic cortex. Herbimycin-treated animals developed spontaneous recurrent seizures, as did control animals, with a similar latency for the appearance of the first seizure and similar seizure frequency. Neo-Timm staining revealed that all animals experiencing SE, regardless of whether or not injected with herbimycin, showed aberrant mossy fiber sprouting in the supragranular region of the dentate gyrus. Herbimycin did not obviously affect neuronal cell death as evaluated in Nissl-stained sections. These results indicate that the PTK blockade achieved with the current dose of herbimycin reduced the acute c-Fos expression but failed to alter the spontaneous seizure frequency or to attenuate the morphological modifications triggered by the SE.     

Dentate granule cell neurogenesis after seizures induced by pentylenetrazol in rats

Epileptic seizures originating from the limbic system have been shown to stimulate the proliferation rate of granule cell precursors in the adult brain, but it is not clear if other type(s) of seizures have the similar effects. This study examined the effects of pentylenetrazol (PTZ)-induced generalized clonic seizures on dentate granule cell neurogenesis in adult rats. Using systemic bromodeoxyuridine (BrdU) to label dividing cells, we studied the proliferation rate of neural precursor cells in the dentate gyrus at various time points after PTZ-induced seizures. The double-label immunofluorescence with confocal microscopy was used to determine the newborn cell phenotypes. Quantitative analysis of BrdU labeling revealed a significant increase in the proliferation rate of neural precursor cells in the dentate gyrus 3, 7, and 14 days after seizures. The number of BrdU-labeled cells in the dentate gyrus returned to baseline levels by 28 days after the initial seizures. Most of newborn cells migrated into the granule cell layer from the subgranular zone, displayed the neuronal phenotype, and developed morphological characteristics of differentiated dentate granule cells. These results indicated that neuron proliferation in the dentate gyrus was enhanced during a time window (3-14 days) after PTZ-induced seizures. Its underlying mechanism is discussed.     

Development of later life spontaneous seizures in a rodent model of hypoxia-induced neonatal seizures

Purpose: To study the development of epilepsy following hypoxia‐induced neonatal seizures in Long‐Evans rats and to establish the presence of spontaneous seizures in this model of early life seizures. Methods: Long‐Evans rat pups were subjected to hypoxia‐induced neonatal seizures at postnatal day 10 (P10). Epidural cortical electroencephalography (EEG) and hippocampal depth electrodes were used to detect the presence of seizures in later adulthood (>P60). In addition, subdermal wire electrode recordings were used to monitor age at onset and progression of seizures in the juvenile period, at intervals between P10 and P60. Timm staining was performed to evaluate mossy fiber sprouting in the hippocampi of P100 adult rats that had experienced neonatal seizures. Key Findings: In recordings made from adult rats (P60–180), the prevalence of epilepsy in cortical and hippocampal EEG recordings was 94.4% following early life hypoxic seizures. These spontaneous seizures were identified by characteristic spike and wave activity on EEG accompanied by behavioral arrest and facial automatisms (electroclinical seizures). Phenobarbital injection transiently abolished spontaneous seizures. EEG in the juvenile period (P10–60) showed that spontaneous seizures first occurred approximately 2 weeks after the initial episode of hypoxic seizures. Following this period, spontaneous seizure frequency and duration increased progressively with time. Furthermore, significantly increased sprouting of mossy fibers was observed in the CA3 pyramidal cell layer of the hippocampus in adult animals following hypoxia‐induced neonatal seizures. Notably, Fluoro‐Jade B staining confirmed that hypoxic seizures at P10 did not induce acute neuronal death. Significance: The rodent model of hypoxia‐induced neonatal seizures leads to the development of epilepsy in later life, accompanied by increased mossy fiber sprouting. In addition, this model appears to exhibit a seizure‐free latent period, following which there is a progressive increase in the frequency of electroclinical seizures.     

Pilocarpine-induced status epilepticus results in mossy fiber sprouting and spontaneous seizures in C57BL/6 and CD-1 mice

Several rodent models are available to study the cellular mechanisms associated with the development of temporal lobe epilepsy (TLE), but few have been successfully transferred to inbred mouse strains commonly used in genetic mutation studies. We examined spontaneous seizure development and correlative axon sprouting in the dentate gyrus of CD-1 and C57BL/6 mice after systemic injection of pilocarpine. Pilocarpine induced seizures and status epilepticus (SE) after systemic injection in both strains, although SE onset latency was greater for C57BL/6 mice. There were also animals of both strains which did not experience SE after pilocarpine treatment. After a period of normal behavior for several days after the pilocarpine treatment, spontaneous tonic-clonic seizures were observed in most CD-1 mice and all C57BL/6 that survived pilocarpine-induced SE. Robust mossy fiber sprouting into the inner molecular layer was observed after 4-8 weeks in mice from both strains which had experienced SE, and cell loss was apparent in the hippocampus. Mossy fiber sprouting and spontaneous seizures were not observed in mice that did not experience a period of SE. These results indicate that pilocarpine induces spontaneous seizures and mossy fiber sprouting in both CD-1 and C57BL/6 mouse strains. Unlike systemic kainic acid treatment, the pilocarpine model offers a potentially useful tool for studying TLE development in genetically modified mice raised on the C57BL/6 background.