Epileptic tolerance is associated with enduring neuroprotection and uncoupling of the relationship between CA3 damage,neuropeptide Y rearrangement and spontaneous seizures following intra-amygdala kainic acid-induced status epilepticus in mice

Brief, non-harmful seizures can activate endogenous protective programmes which render the brain resistant to damage caused by prolonged seizure episodes. Whether protection in epileptic tolerance is long-lasting or influences the subsequent development of epilepsy is uncertain. Presently, we investigated the relationship between hippocampal pathology, neuropeptide Y rearrangement and spontaneous seizures in sham- and seizure-preconditioned mice after status epilepticus induced by intra-amygdala kainate. Seizure-induced neuronal death at 24 h was significantly reduced in the ipsilateral hippocampal CA3 and hilus of tolerance mice compared to sham-preconditioned animals subject to status epilepticus. Damage to the CA3-hilus remained reduced in tolerance mice 21 days post-status. In sham-preconditioned mice subject to status epilepticus correlative statistics showed there was a strong inverse relationship between CA3, but not hilar, neuron counts and the number of spontaneous seizures. A strong positive association was also found between neuropeptide Y score and spontaneous seizure count in these mice. In contrast, there was no significant association between spontaneous seizure count and CA3 neuron loss or neuropeptide Y rearrangement in the tolerance mice. These data show that tolerance-conferred neuroprotection is long-lasting and that tolerance disrupts the normal association between CA3 damage, synaptic rearrangement and occurrence of spontaneous seizures in this model     

Consequences of recurrent seizures during early brain development.

It is well documented that prolonged seizures (status epilepticus) can cause neuronal injury and result in synaptic reorganization in certain brain regions. However, the effect of recurrent, relatively short seizures in young animals on subsequent brain development is not known. To study the consequences of recurrent seizures on the developing brain, we subjected immature rats to a total of 50 flurothyl-induced seizures from postnatal day 11 until day 23. Immunohistochemistry for c-fos was performed to characterize the pattern of neuronal activation following the seizures. Cell counting of dentate granule cells, CA3, CA1, and hilar neurons, using unbiased stereological methods, and the silver impregnation method were used to evaluate neuronal death following the recurrent seizures. Timm and Golgi staining were performed four weeks after the 50th seizure to evaluate the effects of recurrent seizures on synaptic organization. Our results show that recurrent flurothyl-induced seizures progressively increased excitability of the brain, as revealed by a dramatic increase in the extent and intensity of c-fos immunostaining. While no cell loss was detected in the hippocampus with either Cresyl Violet or silver stains, animals experiencing multiple daily seizures developed increased mossy fiber sprouting in both the supragranular layer of the dentate gyrus and the infrapyramidale layer of the CA3 region. Golgi staining confirmed that there was an increase in mossy fibers in the pyramidal cell layer. Our results suggest that serial recurrent seizures in the immature brain can lead to significant changes in mossy fiber distribution even though the seizures do not cause significant hippocampal cell loss.     

Reassessment of the effects of cycloheximide on mossy fiber sprouting and epileptogenesis in the pilocarpine model of temporal lobe epilepsy

A feature of animal models of temporal lobe epilepsy and the human disorder is hippocampal sclerosis and Timm stain in the inner molecular layer (IML) of the dentate gyrus, which represents synaptic reorganization and may be important in epileptogenesis. We reassessed the hypothesis that pre-treatment with cycloheximide (CHX) prevents Timm staining in the IML following pilocarpine (PILO)-induced status epilepticus (a multifocal model of temporal lobe epilepsy), but allows epileptogenesis (i.e., chronic spontaneous seizures) after a latent period. Hippocampal slices from PILO-treated rats without Timm stain in the IML after CHX treatment were hypothesized to lack the electrophysiological abnormalities suggestive of recurrent excitation. The primary experimental groups were as follows: 1) CHX (1 mg/kg) 30-45 min prior to administration of PILO (320 mg/kg ip, 2) only PILO, and 3) only saline (0.5 ml, IP). The CHX pre-treatment significantly decreased the number of rats that responded to PILO with status epilepticus compared to rats that received only PILO. Pre-treatment with CHX did not significantly alter the spontaneous motor seizure rate post-treatment compared to treatment with PILO alone in those animals from each group that developed status epilepticus during PILO treatment. Timm stain in the IML was not significantly different between the PILO- and PILO+CHX-treated rats. Using quantitative methods, CHX did not prevent hilar, CA1, or CA3 neuronal loss compared to the PILO-treated rats. Extracellular responses to hilar stimulation in 30 microM bicuculline and 6 mM [K(+)](o) demonstrated all-or-none bursting in both the CHX+PILO- and PILO-treated rats but not in control rats. Whole cell recordings from granule cells, using glutamate flash photolysis to activate other granule cells, showed that both the CHX+PILO- and PILO-treated rats had excitatory synaptic interactions in the granule cell layer, which were not found after saline treatment. Some rats responded to PILO (with or without CHX pre-treatment) with only one or a few seizures at treatment, and some of these animals (n = 4) demonstrated spontaneous motor seizures within 2 mo after treatment. Timm staining and neuron loss in this group were not clearly different from saline-treated rats. These results suggest that in the PILO model, pre-treatment with CHX does not affect mossy fiber sprouting in the IML of epileptic rats and does not prevent the formation of recurrent excitatory circuits. However, the develoment of spontaneous motor seizures, in a small number of rats, could occur without detectable hippocampal neuron loss or mossy fiber sprouting, as assessed by the Timm stain method.     

A chronic histopathological and electrophysiological analysis of a rodent hypoxic-ischemic brain injury model and its use as a model of epilepsy

Ischemic brain injury is one of the leading causes of epilepsy in the elderly, and there are currently no adult rodent models of global ischemia, unilateral hemispheric ischemia, or focal ischemia that report the occurrence of spontaneous motor seizures following ischemic brain injury. The rodent hypoxic-ischemic (H-I) model of brain injury in adult rats is a model of unilateral hemispheric ischemic injury. Recent studies have shown that an H-I injury in perinatal rats causes hippocampal mossy fiber sprouting and epilepsy. These experiments aimed to test the hypothesis that a unilateral H-I injury leading to severe neuronal loss in young-adult rats also causes mossy fiber sprouting and spontaneous motor seizures many months after the injury, and that the mossy fiber sprouting induced by the H-I injury forms new functional recurrent excitatory synapses. The right common carotid artery of 30-day old rats was permanently ligated, and the rats were placed into a chamber with 8% oxygen for 30 min. A quantitative stereologic analysis revealed that the ipsilateral hippocampus had significant hilar and CA1 pyramidal neuronal loss compared with the contralateral and sham-control hippocampi. The septal region from the ipsilateral and contralateral hippocampus had small but significantly increased amounts of Timm staining in the inner molecular layer compared with the sham-control hippocampi. Three of 20 lesioned animals (15%) were observed to have at least one spontaneous motor seizure 6-12 months after treatment. Approximately 50% of the ipsilateral and contralateral hippocampal slices displayed abnormal electrophysiological responses in the dentate gyrus, manifest as all-or-none bursts to hilar stimulation. This study suggests that H-I injury is associated with synaptic reorganization in the lesioned region of the hippocampus, and that new recurrent excitatory circuits can predispose the hippocampus to abnormal electrophysiological activity and spontaneous motor seizures.     

Expression of synaptopodin, an actin-associated protein, in the rat hippocampus after limbic epilepsy

Synaptopodin, a 100 kD protein, associated with the actin cytoskeleton of the postsynaptic density and dendritic spines, is thought to play a role in modulating actin-based shape and motility of dendritic spines during formation or elimination of synaptic contacts. Temporal lobe epilepsy in humans and in rats shows neuronal damage, aberrant sprouting of hippocampal mossy fibers and subsequent synaptic remodeling processes. Using kainic acid (KA) induced epilepsy in rats, the postictal hippocampal expression of synaptopodin was analyzed by in situ hybridization (ISH) and immunohistochemistry. Sprouting of mossy fibers was visualized by a modified Timm's staining. ISH showed elevated levels of Synaptopodin mRNA in perikarya of CA3 principal neurons, dentate granule cells and in surviving hilar neurons these levels persisted up to 8 weeks after seizure induction. Synaptopodin immunoreactivity in the dendritic layers of CA3, in the hilus and in the inner molecular layer of the dentate gyrus (DG) was initially reduced. Eight weeks after KA treatment Synaptopodin protein expression returned to control levels in dendritic layers of CA3 and in the entire molecular layer of the DG. The recovery of protein expression was accompanied by simultaneous supra- and infragranular mossy fiber sprouting. Postictal upregulation of Synaptopodin mRNA levels in target cell populations of limbic epilepsy-elicited damage and subsequent Synaptopodin protein expression largely co-localized with remodeling processes as demonstrated by mossy fiber sprouting. It may thus represent a novel postsynaptic molecular correlate of hippocampal neuroplasticity.     

Activity-dependent changes in synaptophysin immunoreactivity in hippocampus,piriform cortex,and entorhinal cortex of the rat

Synaptophysin, an integral membrane glycoprotein of synaptic vesicles, has been widely used to investigate synaptogenesis in both animal models and human patients. Kindling is an experimental model of complex partial seizures with secondary generalization, and a useful model for studying activation-induced neural growth in adult systems. Many studies using Timm staining have shown that kindling promotes sprouting in the mossy fiber pathway of the dentate gyrus. In the present study, we used synaptophysin immunohistochemistry to demonstrate activation-induced neural sprouting in non-mossy fiber cortical pathways in the adult rat. We found a significant kindling-induced increase in synaptophysin immunoreactivity in the stratum radiatum of CA1 and stratum lucidum/radiatum of CA3, the hilus, the inner molecular layer of the dentate gyrus, and layer II/III of the piriform cortex, but no significant change in layer II/III of the entorhinal cortex, 4 weeks after the last kindling stimulation. We also found that synaptophysin immunoreactivity was lowest in CA3 near the hilus and increased with increasing distance from the hilus, a reverse pattern to that seen with Timm stains in stratum oriens following kindling. Furthermore, synaptophysin immunoreactivity was lowest in dorsal and greatest in ventral sections of both CA3 and dentate gyrus in both kindled and non-kindled animals. This demonstrates that different populations of sprouting axons are labeled by these two techniques, and suggests that activation-induced sprouting extends well beyond the hippocampal mossy fiber system.     

Mouse models of human KCNQ2 and KCNQ3 mutations for benign familial neonatal convulsions show seizures and neuronal plasticity without synaptic reorganization

The childhood epilepsy syndrome of benign familial neonatal convulsions (BFNC) exhibits the remarkable feature of clinical remission within a few weeks of onset and a favourable prognosis, sparing cognitive abilities despite persistent expression of the mutant KCNQ2 or KCNQ3 potassium channels throughout adulthood. To better understand such dynamic neuroprotective plasticity within the developing brain, we introduced missense mutations that underlie human BFNC into the orthologous murine Kcnq2 (Kv7.2) and Kcnq3 (Kv7.3) genes. Mutant mice were examined for altered thresholds to induced seizures, spontaneous seizure characteristics, hippocampal histology, and M-current properties of CA1 hippocampal pyramidal neurons. Adult Kcnq2 ^A306T/+ and Kcnq3 ^G311V/+ heterozygous knock-in mice exhibited reduced thresholds to electrically induced seizures compared to wild-type littermate mice. Both Kcnq2 ^A306T/A306T and Kcnq3 ^G311V/G311V homozygous mutant mice exhibited early onset spontaneous generalized tonic-clonic seizures concurrent with a significant reduction in amplitude and increased deactivation kinetics of the neuronal M-current. Mice had recurrent seizures into adulthood that triggered molecular plasticity including ectopic neuropeptide Y (NPY) expression in granule cells, but without hippocampal mossy fibre sprouting or neuronal loss. These novel knockin mice recapitulate proconvulsant features of the human disorder yet show that inherited M-current defects spare granule cells from reactive changes in adult hippocampal networks. The absence of seizure-induced pathology found in these epileptic mouse models parallels the benign neurodevelopmental cognitive profile exhibited by the majority of BFNC patients.     

海人酸诱导癫痫大鼠齿状回神经肽Y能苔状纤维侧枝发芽

目的：探讨海马苔状纤维侧枝发芽与癫痫发作敏感性形成的关系。材料与方法：在海人酸诱发大鼠出现癫痫发作后,采用免疫组织化学染色法观察大鼠海马齿状回内神经肽Ｙ能纤维的异常发芽。结果：首次证实在癫痫发作后７天时,在海马齿状回内分子层就已出现神经肽Ｙ能纤维的异常增生。结论：这可能是对癫痫发作后齿状回门区神经肽Ｙ能神经元缺失的一种代偿性变化。     

Protective effects of mossy fiber lesions against kainic acid-induced seizures and neuronal degeneration

The effects of a hippocampal mossy fiber lesion have been determined on neuronal degeneration and limbic seizures provoked by the subsequent intracerebroventricular administration of kainic acid to unanesthetized rats. Mossy fiber lesions were made either by transecting this pathway unilaterally or by destroying the dentate granule cells unilaterally or bilaterally with colchicine. All control rats eventually developed status epilepticus and each temporally discrete seizure that preceded status epilepticus was recorded from the hippocampus ipsilateral to the kainic acid infusion before the contralateral hippocampus. A mossy fiber lesion of the ipsilateral hippocampus prevented the development of status epilepticus in 26% of subjects and in 52% of subjects seizures were recorded from the contralateral hippocampus before the ipsilateral hippocampus. Unlike electrographic records from other treatment groups, those from rats which had received a bilateral colchicine lesion exhibited no consistent pattern indicative of seizure propagation from one limbic region to another. A bilateral, but not a unilateral, mossy fiber lesion also dramatically attenuated the behavioral expression of the seizures. Regardless of its effects on kainic acid-induced electrographic and behavioral seizures, a mossy fiber lesion always substantially reduced or completely prevented the degeneration of ipsilateral hippocampal CA3-CA4 neurons. This protective effect was specific for those hippocampal neurons deprived of mossy fiber innervation. Neurons in other regions of the brain were protected from degeneration only when the mossy fiber lesion also prevented the development of electrographic status epilepticus. These results suggest that the hippocampal mossy fibers constitute an important, though probably not an obligatory, link in the circuit responsible for the spread of kainic acid seizures. Degeneration of CA3-CA4 neurons appears to depend upon (1) the duration of hippocampal seizure activity and (2) an as yet undefined influence of or interaction with the mossy fiber projection which enhances the neurodegenerative effect of the seizures.     

Bcl-w protects hippocampus during experimental status epilepticus

Experimentally evoked seizures can activate the intrinsic mitochondrial cell death pathway, components of which are modulated in the hippocampus of patients with temporal lobe epilepsy. Bcl-2 family proteins are critical regulators of mitochondrial dysfunction, but their significance in this setting remains primarily untested. Presently, we investigated the mitochondrial pathway and role of anti-apoptotic Bcl-2 proteins using a mouse model of seizure-induced neuronal death. Status epilepticus was evoked in mice by intra-amygdala kainic acid, causing cytochrome c release, processing of caspases 9 and 7, and death of ipsilateral hippocampal pyramidal neurons. Seizures caused a rapid decline in hippocampal Bcl-w levels not seen for either Bcl-2 or Bcl-xl. To test whether endogenous Bcl-w was functionally significant for neuronal survival, we investigated hippocampal injury after seizures in Bcl-w-deficient mice. Seizures induced significantly more hippocampal CA3 neuronal loss and DNA fragmentation in Bcl-w-deficient mice compared with wild-type mice. Quantitative electroencephalography analysis also revealed that Bcl-w-deficient mice display a neurophysiological phenotype whereby there was earlier polyspike seizure onset. Finally, we detected higher levels of Bcl-w in hippocampus from temporal lobe epilepsy patients compared with autopsy controls. These data identify Bcl-w as an endogenous neuroprotectant that may have seizure-suppressive functions.     

Mice deficient for the extracellular matrix glycoprotein tenascin-r show physiological and structural hallmarks of increased hippocampal excitability, but no increased susceptibility to seizures in the pilocarpine model of epilepsy

Recognition molecules provide important cues for neuronal survival, axonal fasciculation, axonal pathfinding, synaptogenesis, synaptic plasticity, and regeneration. Our previous studies revealed a link between perisomatic inhibition and the extracellular matrix glycoprotein tenascin-R (TN-R). Therefore, we here studied neuronal excitability and epileptic susceptibility in mice constitutively deficient in TN-R. In vitro analysis of populational spikes in hippocampal slices of TN-R-deficient mice revealed a significant increase in multiple spikes in the CA1 region, as compared with wild-type mice. This difference between genotypes was only partially reduced after blockade of GABA(A) receptors with picrotoxin, indicating a deficit in GABAergic inhibition and an increase in intrinsic excitability of CA1 pyramidal cells in TN-R-deficient mice. Using a battery of immunohistochemical markers and histological stainings, we were able to identify two abnormalities in the hippocampus of TN-R-deficient mice possibly related to increased excitability: the high number of glial fibrillary acidic protein-positive astrocytes and low number of calretinin-positive interneurons in the CA1 and CA3 regions. In order to test whether the revealed abnormalities give rise to increased susceptibility to seizures in TN-R-deficient mice, we used the pilocarpine model of epilepsy. No genotype-specific differences were found with regard to the time-course of pilocarpine-induced and spontaneous seizures, neuronal cell loss, aberrant sprouting and distribution of synaptic and inhibitory interneuron markers. However, pilocarpine-induced astrogliosis and reduction in calretinin-positive interneurons were less pronounced in TN-R mutants, thereby resulting in an occlusion of effects induced by TN-R deficiency and pilocarpine. Thus, TN-R-deficient mutants show several electrophysiological and morphological hallmarks of increased neuronal excitability, which, however, do not give rise to more accelerated or severe epileptogenesis in the pilocarpine model of epilepsy.     

Evolution of hippocampal epileptic activity during the development of hippocampal sclerosis in a mouse model of temporal lobe epilepsy

Unilateral intrahippocampal injection of kainic acid in adult mice reproduces most of the morphological characteristics of hippocampal sclerosis (neuronal loss, gliosis, reorganization of neurotransmitter receptors, mossy fiber sprouting, granule cell dispersion) observed in patients with temporal lobe epilepsy. Whereas some neuronal loss is observed immediately after the initial status epilepticus induced by kainate treatment, most reorganization processes develop progressively over a period of several weeks. The aim of this study was to characterize the evolution of seizure activity in this model and to assess its pharmacological reactivity to classical antiepileptic drugs.Intrahippocampal electroencephalographic recordings showed three distinct phases of paroxystic activity following unilateral injection of kainic acid (1 nmol in 50 nl) into the dorsal hippocampus of adult mice: (i) a non-convulsive status epilepticus, (ii) a latent phase lasting approximately 2 weeks, during which no organized activity was recorded, and (iii) a phase of chronic seizure activity with recurrent hippocampal paroxysmal discharges characterized by high amplitude sharp wave onset. These recurrent seizures were first seen about 2 weeks post-injection. They were limited to the injected area and were not observed in the cerebral cortex, contralateral hippocampus or ipsilateral amygdala. Secondary propagation to the contralateral hippocampus and to the cerebral cortex was rare. In addition hippocampal paroxysmal discharges were not responsive to acute carbamazepine, phenytoin, or valproate treatment, but could be suppressed by diazepam.Our data further validate intrahippocampal injection of kainate in mice as a model of temporal lobe epilepsy and suggest that synaptic reorganization in the lesioned hippocampus is necessary for the development of organized recurrent seizures.     

Acupuncture suppresses kainic acid-induced neuronal death and inflammatory events in mouse hippocampus

The administration of kainic acid (KA) causes seizures and produces neurodegeneration in hippocampal CA3 pyramidal cells. The present study investigated a possible role of acupuncture in reducing hippocampal cell death and inflammatory events, using a mouse model of kainic acid-induced epilepsy. Male C57BL/6 mice received acupuncture treatments at acupoint HT8 or in the tail area bilaterally once a day for 2 days and again immediately after an intraperitoneal injection of KA (30 mg/kg). HT8 is located on the palmar surface of the forelimbs, between the fourth and fifth metacarpal bones. Twenty-four hours after the KA injection, neuronal cell survival, the activations of microglia and astrocytes, and mRNA expression of two proinflammatory cytokines, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), were measured in the hippocampus. Acupuncture stimulation at HT8, but not in the tail area, significantly reduced the KA-induced seizure, neuron death, microglial and astrocyte activations, and IL-1β mRNA expression in the hippocampus. The acupuncture stimulation also decreased the mRNA expression of TNF-α, but it was not significant. These results indicate that acupuncture at HT8 can inhibit hippocampal cell death and suppress KA-induced inflammatory events, suggesting a possible role for acupuncture in the treatment of epilepsy.     

正常成年Wistar大鼠海马结构苔藓纤维的分布

用 Timm显影染色方法 ,研究正常 Wistar大鼠海马结构苔藓纤维的分布。结果苔藓纤维主要集中在门区和 CA3区透明层 ,但在 CA3区始层所有大鼠都多少不等地有银染颗粒即苔藓纤维终扣分布 ,8/2 0只大鼠甚至达到 2至 3级 ,呈稀疏到浓密条带状 ,此点与既往的报道不同 ;还有 13 /2 0只大鼠在齿状回有较多苔藓纤维呈条束状由门区发支穿越颗粒细胞层     

Effect of PACAP on MAP kinases, Akt and cytokine expressions in rat retinal hypoperfusion

Immunohistochemical studies were performed to analyze the expressional changes in hippocampal synaptic vesicle protein 2A (SV2A) following pentylenetetrazole (PTZ) kindling. Repeated treatments of mice with sub-convulsive PTZ (40 mg/kg, i.p.) for 15 days progressively enhanced seizure susceptibility and induced clonic convulsions in most animals examined. Topographical analysis of hippocampal SV2A-immunoreactivity revealed that SV2A was densely expressed in the hilar region of the dentate gyrus, stratum lucidum of the CA3 field and around the periphery of CA3 pyramidal neurons. PTZ kindling region-specifically increased SV2A expression in the dentate hilus without affecting that in the stratum lucidum or the pyramidal cell layer of the CA3 field. Confocal laser microscopic analysis using PTZ-kindled mice illustrated that most SV2A was co-expressed with glutamic acid decarboxylase 67 in the cell bodies and dendrites of hilar interneurons. However, SV2A-immunoreactivity was negligibly observed in the hilar glutamatergic nerve terminals (mossy fibers) probed with the anti-vesicular glutamate transporter 1 antibody. The present study suggests that SV2A specifically regulates hilar GABAergic neurotransmission in the kindled hippocampus probably as a compensatory or prophylactic mechanism against kindling epileptogenesis.     

Sprouting and synaptic reorganization in the subiculum and CA1 region of the hippocampus in acute and chronic models of partial-onset epilepsy

Repeated seizures induce permanent alterations in the hippocampal circuitry in experimental models and patients with intractable temporal lobe epilepsy (TLE). Most studies have concentrated their attention on seizure-induced reorganization of the mossy fiber pathway. The present study examined the projection pathway of the CA1 pyramidal neurons to the subiculum, which is the output of the hippocampal formation in five models of TLE. We examined the laminar pattern of Timm's histochemistry in the stratum lacunosum-moleculare of CA1 in three acute and two chronic models of TLE: intraventricular kainic acid (KA), systemic KA, systemic pilocarpine, chronic electric kindling and chronic i.p. pentylenetetrazol. The laminar pattern of Timm histochemistry in the stratum moleculare of CA1 was permanently remodeled in epileptic models suggesting sprouting of Timm containing terminals from the adjacent stratum lacunosum. Ultrastructural examination confirmed that Timm granules were localized in synaptic terminals. As the source of Timm-labeled terminals in this region was not known, sodium selenite, a selective retrograde tracer for zinc-containing terminals, was iontophoretically injected in vivo in rats exposed to systemic pilocarpine, systemic KA or chronic pentylenetetrazol. The normal projection of CA1 pyramidal neurons to the subiculum is topographically organized in a lamellar fashion. In normal rats, the extent of the injection site (terminals) and the retrogradely labeled pyramidal neurons (cell soma) corresponded to the same number of lamellas. In epileptic rats, the retrograde labeling extended 42-67% farther than the normal dorso-ventral extent including lamellas above and below the expected. This is direct evidence for sprouting of CA1 pyramidal axons into the subiculum and stratum lacunosum-moleculare of the CA1 region confirming the alterations of the laminar pattern of Timm's histochemistry. Sprouting of the CA1 projection to subiculum across hippocampal lamellas might lead to translamellar hyperexcitability, and to amplification and synchronization of epileptic discharges in the output gate of the hippocampal formation.     

Mouse strain differences in kainic acid sensitivity, seizure behavior, mortality, and hippocampal pathology

Genetic influences contribute to susceptibility to seizures and to excitotoxic injury, but it is unclear if/how these susceptibilities are linked. This study assessed the impact of genetic background on mouse strain seizure susceptibility, seizure phenotype, mortality, and hippocampal histopathology. A subcutaneous (s.c.) kainic acid multiple injection protocol was developed. Five mouse strains were tested: a and b) C57BL/6J and 129/SvJ, strains commonly used in gene targeting experiments; c) C3HeB/FeJ, a strain with reported sensitivity to the convulsant effects of kainic acid (KA); d) 129/SvEms, a strain reportedly susceptible to hippocampal excitotoxic cell death; and e) a mixed genetic background strain (129/SvJXC57BL/6J) from which targeted gene deletion experiments have been carried out. Histopathological features were examined at early (7-10 day), delayed (2-4 month), and late (6-13 month) time points.Mouse background strains can be genetically segregated based on excitotoxin sensitivity, seizure phenotype, mortality, and hippocampal histopathology. When injected with KA, C3HeB/FeJ and C57BL/6J strains were resistant to cell death and synaptic reorganization despite severe behavioral seizures, while 129/SvEms mice developed marked pyramidal cell loss and mossy fiber sprouting despite limited seizure activity. The mixed background 129/SvJXC57BL/6J group exhibited features of both parental strains. In the mouse strains tested, the duration or severity of seizure activity was not predictive of subsequent hippocampal pyramidal cell death and/or synaptic reorganization. Unlike rats, mice exhibiting prolonged high-grade KA-induced seizure activity did not develop subsequent spontaneous behavioral seizures.     

Temporal ontogeny of circuit activation prior to the onset of seizure susceptibility in EL/Suz mice

The EL/Suz (EL) mouse is a model of multifactorial temporal lobe epilepsy in which seizures begin around 90 days of age, but can be hastened through increased exposure to human handling. In order to better understand seizure etiology in this mouse strain relative to seizure-resistant control mice, the present study examined region-specific neuronal activation in response to non-seizure-inducing handling implemented before the onset of seizure susceptibility. Immediate-early gene (cFos) expression emerged in EL mice by postnatal day (PND) 21 in the primary motor cortex, progressed to the locus ceruleus and prefrontal cortex by PND 35, and appeared in the hippocampus and amygdala by PND 70, as mice neared the age of onset for seizure susceptibility. Thus, mirroring the pattern observed during the course of a seizure, specific brain regions were differentially recruited to a neural network for seizure predisposition before the onset of seizure susceptibility. This developmental pattern of early and transient neural activation represents an important window for the study of causal mechanisms of seizure susceptibility following exposure to environmental triggers.     

Hippocampal cell proliferation and epileptogenesis after audiogenic kindling are not accompanied by mossy fiber sprouting or Fluoro-Jade staining

Repetitive sound-induced seizures, known as audiogenic kindling (AK), gradually induce the transference of epileptic activity from brainstem to forebrain structures along with behavioral changes. The aim of our work was to correlate the behavioral changes observed during the AK with possible alterations in neuronal proliferation, cell death, hippocampal mossy fiber sprouting and in the EEG pattern of Wistar audiogenic rats, a genetically susceptible strain from our laboratory.Susceptible and non-susceptible animals were submitted to repeated sound stimulations for 14-16 days and hippocampal mitotic activity was studied through the incorporation of bromodeoxyuridine (BrdU). Cell death and mossy fiber sprouting were assessed, respectively, by using Fluoro-Jade and Timm staining, 2 and 32 days after the last kindling stimulation. In addition, we used immunofluorescent double labeling for a glial and a mitotic marker to evaluate newly born cell identity. Some animals had hippocampus and amygdala electrodes for EEG recordings.Our results show that kindled animals with 6-11 generalized limbic seizures (class IV-V) had increased cell proliferation in the dentate gyrus when compared with animals with zero or one to three seizures. BrdU-positive cells labeled on day 2 and on day 32 were both GFAP negative. In the later group, rounded and well-defined BrdU-positive/GFAP-negative nuclei were seen in different portions of the granule cell layer. We did not observe any Fluoro-Jade or differential Timm staining in kindled animals at both killing times. However, EEG recordings showed intense epileptic activity in the hippocampus and amygdala of all animals with limbic seizures.Therefore, our data indicate that AK-induced limbic epileptogenicity is able to increase the hippocampal mitotic rate, even though it does not seem to promote neuronal death or mossy fiber sprouting in the supragranular layer of the dentate gyrus.     

Zinc chelation during non-lesioning overexcitation results in neuronal death in the mouse hippocampus

In the hippocampus, chelatable zinc is accumulated in vesicles of glutamatergic presynaptic terminals, abounding specially in the mossy fibers, from where it is released with activity and can exert a powerful inhibitory action upon N-methyl-D-aspartate receptors. Zinc is therefore in a strategic situation to control overexcitation at the zinc-rich excitatory synapses, and consequently zinc removal during high activity might result in excitotoxic neuronal damage. We analyzed the effect of zinc chelation with sodium dietyldithiocarbamate under overexcitation conditions induced by non-lesioning doses of kainic acid in the mouse hippocampus, to get insight into the role of zinc under overexcitation. Swiss male mice were injected with kainic acid (15 mg/kg, i.p.) 15 min prior to sodium dietyldithiocarbamate (150 mg/kg, i.p.), and left to survive for 6 h, 1 day, 4 days, or 7 days after the treatment. Cell damage was analyzed with the hematoxylin-eosin and acid fuchsin stainings. Neither control animals treated only with kainic acid nor those treated only with sodium dietyldithiocarbamate suffered seizures or neuronal damage. By contrast, the kainic acid+sodium dietyldithiocarbamate-treated animals showed convulsive behavior and cell death involving the hilus, CA3, and CA1 regions. Pretreatment with the N-methyl-D-aspartate receptor antagonist MK801 (1 mg/kg, i.p.) completely prevented neuronal damage. Experiments combining different doses of sodium dietyldithiocarbamate and kainic acid with different administration schedules demonstrated that the overlap of zinc chelation and overexcitation is necessary to trigger the observed effects. Moreover, the treatment with a high dose of sodium dietyldithiocarbamate (1000 mg/kg), which produced a complete bleaching of the Timm staining for approximately 12 h, highly increased the sensitivity of animals to kainic acid. Altogether, our results indicate that the actions of sodium dietyldithiocarbamate are based on a reduction of zinc levels, which under overexcitation conditions induce seizures and neuronal damage. These findings fully support a protective role for synaptically released zinc during high neuronal activity, most probably mediated by its inhibitory actions on N-methyl-D-aspartate receptors, and argue against a direct action of synaptic zinc on the observed neuronal damage.