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Alterations in the balance of inhibitory and excitatory synaptic transmission have been implicated in the pathogenesis of neurological disorders such as epilepsy. Eukaryotic elongation factor 2 kinase (eEF2K) is a highly regulated, ubiquitous kinase involved in the control of protein translation. Here, we show that eEF2K activity negatively regulates GABAergic synaptic transmission. Indeed, loss of eEF2K increases GABAergic synaptic transmission by upregulating the presynaptic protein Synapsin 2b and α5-containing GABAA receptors and thus interferes with the excitation/inhibition balance. This cellular phenotype is accompanied by an increased resistance to epilepsy and an impairment of only a specific hippocampal-dependent fear conditioning. From a clinical perspective, our results identify eEF2K as a potential novel target for antiepileptic drugs, since pharmacological and genetic inhibition of eEF2K can revert the epileptic phenotype in a mouse model of human epilepsy., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Purpose: To investigate the mechanism of the anti-epileptic effect of Ganoderma lucidum polysaccharides (GLP), the changes of intracellular calcium and CaMK II α expression in a model of epileptic neurons were investigated., Method: Primary hippocampal neurons were divided into: 1) Control group, neurons were cultured with Neurobasal medium, for 3 hours; 2) Model group I: neurons were incubated with Mg(2+) free medium for 3 hours; 3) Model group II: neurons were incubated with Mg(2+) free medium for 3 hours then cultured with the normal medium for a further 3 hours; 4) GLP group I: neurons were incubated with Mg(2+) free medium containing GLP (0.375 mg/ml) for 3 hours; 5) GLP group II: neurons were incubated with Mg(2+) free medium for 3 hours then cultured with a normal culture medium containing GLP for a further 3 hours. The CaMK II α protein expression was assessed by Western-blot. Ca(2+) turnover in neurons was assessed using Fluo-3/AM which was added into the replacement medium and Ca(2+) turnover was observed under a laser scanning confocal microscope., Results: The CaMK II α expression in the model groups was less than in the control groups, however, in the GLP groups, it was higher than that observed in the model group. Ca(2+) fluorescence intensity in GLP group I was significantly lower than that in model group I after 30 seconds, while in GLP group II, it was reduced significantly compared to model group II after 5 minutes., Conclusion: GLP may inhibit calcium overload and promote CaMK II α expression to protect epileptic neurons.

Calcium/calmodulin-dependent protein kinase type II (CaMKII) is a highly abundant serine/threonine kinase comprising a significant fraction of total protein in mammalian forebrain and forming a major component of the postsynaptic density. CaMKII is essential for certain forms of synaptic plasticity and memory consolidation and this is mediated through substrate binding and intramolecular phosphorylation of holoenzyme subunits. CaMKII is multifunctional; it targets a variety of cellular substrates, and this diversity depends on holoenzyme subunit composition. CaMKII comprises homooligomeric and heterooligomeric complexes generated from four subunits (α, β, δ, and γ) encoded by separate genes that are further expanded by extensive alternative splicing to more than 30 different isoforms. Much attention has been paid to understanding the regulation of CaMKII function through its structural diversity and/or substrate specificity. However, given the importance of subunit composition to holoenzyme activity, it is likely that specificity of cellular expression of CaMKII isoforms also plays a major role in regulation of enzyme function. Herein we review the cellular colocalization of CaMKII isoforms with special regard to the cell-type specificity of isoform expression in brain. In addition, we highlight the remarkable specificity of subcellular localization by the CaMKIIα isoform. In addition, we discuss the role that this cellular specificity of expression might play in propagating the type of recurrent neuronal activity associated with disorders such as temporal lobe epilepsy., (Wiley Periodicals, Inc. © 2012 International League Against Epilepsy.)

Disruption of calcium homeostasis in epileptic cells is characterized by both short- and long-term perturbations of Ca(2+) buffering systems. Along with the Na(+)/Ca(2+) exchanger, the plasma membrane Ca(2+)-ATPase (PMCA) plays an important role in excitable cells. The involvement of PMCAs in epileptogenesis has primarily been studied in brief intervals after various stimuli; however, the specific contribution of this molecule to epileptogenesis is not yet fully understood. Our aim has been to investigate whether PMCA expression in the chronic stages of epilepsy is altered. Through an interdisciplinary approach, involving whole-cell recordings and real-time reverse transcriptase-polymerase chain reaction, we have shown that epileptic neurons in our preparation consistently show changes in electrical properties during the period of chronic epilepsy. These changes included increased spike frequency, altered resting membrane potential and changes in passive membrane properties. Following these observations, which indicate an altered excitability in the epileptic cells studied, PMCA mRNA transcripts were studied. It was found that while PMCA1 transcripts are significantly increased one month following the pilocarpine epileptogenic stimulus, PMCA3, an isoform important in excitable tissues, was significantly, decreased. These findings suggest that, in the long-term, a slow PMCA (PMCA1) plays a role in the reestablishment of a new calcium homeostasis attained by epileptic cells. Overall, this phenomenon points out the fact that in seizure disorders, changes that take place in the balance of the different molecules and their isoforms in charge of maintaining neuronal calcium homeostasis, are fundamental in the survival of affected cells.

Tay-Sachs disease is a severe lysosomal disorder caused by mutations in the HexA gene coding for the α-subunit of lysosomal β-hexosaminidase A, which converts G(M2) to G(M3) ganglioside. Hexa(-/-) mice, depleted of β-hexosaminidase A, remain asymptomatic to 1 year of age, because they catabolise G(M2) ganglioside via a lysosomal sialidase into glycolipid G(A2), which is further processed by β-hexosaminidase B to lactosyl-ceramide, thereby bypassing the β-hexosaminidase A defect. Since this bypass is not effective in humans, infantile Tay-Sachs disease is fatal in the first years of life. Previously, we identified a novel ganglioside metabolizing sialidase, Neu4, abundantly expressed in mouse brain neurons. Now we demonstrate that mice with targeted disruption of both Neu4 and Hexa genes (Neu4(-/-);Hexa(-/-)) show epileptic seizures with 40% penetrance correlating with polyspike discharges on the cortical electrodes of the electroencephalogram. Single knockout Hexa(-/-) or Neu4(-/-) siblings do not show such symptoms. Further, double-knockout but not single-knockout mice have multiple degenerating neurons in the cortex and hippocampus and multiple layers of cortical neurons accumulating G(M2) ganglioside. Together, our data suggest that the Neu4 block exacerbates the disease in Hexa(-/-) mice, indicating that Neu4 is a modifier gene in the mouse model of Tay-Sachs disease, reducing the disease severity through the metabolic bypass. However, while disease severity in the double mutant is increased, it is not profound suggesting that Neu4 is not the only sialidase contributing to the metabolic bypass in Hexa(-/-) mice., Competing Interests: The authors have declared that no competing interests exist.

Matrix metalloproteases (MMPs) degrade or modify extracellular matrix or membrane-bound proteins in the brain. MMP-2 and MMP-9 are activated by treatments that result in a sustained neuronal depolarization and are thought to contribute to neuronal death and structural remodeling. At the synapse, MMP actions on extracellular proteins contribute to changes in synaptic efficacy during learning paradigms. They are also activated during epileptic seizures, and MMP-9 has been associated with the establishment of aberrant synaptic connections after neuronal death induced by kainate treatment. It remains unclear whether MMPs are activated by epileptic activities that do not induce cell death. Here we examine this point in two animal models of epilepsy that do not involve extensive cell damage. We detected an elevation of MMP-9 enzymatic activity in cortical regions of secondary generalization after focal seizures induced by 4-aminopyridine (4-AP) application in rats. Pro-MMP-9 levels were also higher in Wistar Glaxo Rijswijk (WAG/Rij) rats, a genetic model of generalized absence epilepsy, than they were in Sprague-Dawley rats, and this elevation was correlated with diurnally occurring spike-wave-discharges in WAG/Rij rats. The increased enzymatic activity of MMP-9 in these two different epilepsy models is associated with synchronized neuronal activity that does not induce widespread cell death. In these epilepsy models MMP-9 induction may therefore be associated with functions such as homeostatic synaptic plasticity rather than neuronal death., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The mechanism of status epilepticus-induced neuronal death in the immature brain is not fully understood. In the present study, we examined the contribution of caspases in our lithium-pilocarpine model of status epilepticus in 14 days old rat pups. In CA1, upregulation of caspase-8, but not caspase-9, preceded caspase-3 activation in morphologically necrotic cells. Pretreatment with a pan-caspase inhibitor provided neuroprotection, showing that caspase activation was not an epiphenomenon but contributed to neuronal necrosis. By contrast, upregulation of active caspase-9 and caspase-3, but not caspase-8, was detected in apoptotic dentate gyrus neurons, which were immunoreactive for doublecortin and calbindin-negative, two features of immature neurons. These results suggest that, in cells which are aligned in series as parts of the same excitatory hippocampal circuit, the same seizures induce neuronal death through different mechanisms. The regional level of neuronal maturity may be a determining factor in the execution of a specific death program.

Extracellular signal-regulated kinase (ERK) is highly sensitive to regulation by neuronal activity and is critically involved in several forms of synaptic plasticity. These features suggested that alterations in ERK signaling might occur in epilepsy. Previous studies have described increased ERK phosphorylation immediately after the induction of severe seizures, but patterns of ERK activation in epileptic animals during the chronic period have not been determined. Thus, the localization and abundance of phosphorylated extracellular signal-regulated kinase (pERK) were examined in a pilocarpine model of recurrent seizures in C57BL/6 mice during the seizure-free period and at short intervals after spontaneous seizures. Immunolabeling of pERK in control animals revealed an abundance of distinctly-labeled neurons within the hippocampal formation. However, in pilocarpine-treated mice during the seizure-free period, the numbers of pERK-labeled neurons were substantially decreased throughout much of the hippocampal formation. Double labeling with a general neuronal marker suggested that the decrease in pERK-labeled neurons was not due primarily to cell loss. The decreased ERK phosphorylation in seizure-prone animals was interpreted as a compensatory response to increased neuronal excitability within the network. Nevertheless, striking increases in pERK labeling occurred at the time of spontaneous seizures and were evident in large populations of neurons at very short intervals (as early as 2 min) after detection of a behavioral seizure. These findings suggest that increased pERK labeling could be one of the earliest immunohistochemical indicators of neurons that are activated at the time of a spontaneous seizure.

Brain cell vulnerability to neurologic insults varies greatly, depending on their neuronal subpopulation. Among cells that survive a pathological insult such as ischemia or brain trauma, some may undergo morphological and/or biochemical changes that could compromise brain function. We previously reported that surviving cortical GABAergic neurons exposed to glutamate in vitro displayed an NMDA receptor (NMDAR)-mediated alteration in the levels of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD65/67) [Monnerie, H., Le Roux, P., 2007. Reduced dendrite growth and altered glutamic acid decarboxylase (GAD) 65- and 67-kDa isoform protein expression from mouse cortical GABAergic neurons following excitotoxic injury in vitro. Exp. Neurol. 205, 367-382]. In this study, we examined the mechanisms by which glutamate excitotoxicity caused a change in cortical GABAergic neurons' GAD protein levels. Removing extracellular calcium prevented the NMDAR-mediated decrease in GAD protein levels, measured using Western blot techniques, whereas inhibiting calcium entry through voltage-gated calcium channels had no effect. Glutamate's effect on GAD protein isoforms was significantly attenuated by preincubation with the cysteine protease inhibitor N-Acetyl-L-Leucyl-L-Leucyl-L-norleucinal (ALLN). Using class-specific protease inhibitors, we observed that ALLN's effect resulted from the blockade of calpain and cathepsin protease activities. Cell-free proteolysis assay confirmed that both proteases were involved in glutamate-induced alteration in GAD protein levels. Together these results suggest that glutamate-induced excitotoxic stimulation of NMDAR in cultured cortical neurons leads to altered GAD protein levels from GABAergic neurons through intracellular calcium increase and protease activation including calpain and cathepsin. Biochemical alterations in surviving cortical GABAergic neurons in various disease states may contribute to the altered balance between excitation and inhibition that is often observed after injury.

Homeostatic plasticity mechanisms stabilize the activity of a neuron or neuronal circuit during prolonged periods of increased network activity and have been proposed to function in the prevention of epilepsy. How homeostatic plasticity is achieved at the molecular level during hyperactivity states in general, and during epileptiform activity in particular, is unclear. Using organotypic hippocampal slice cultures as a model system, we found that the protein kinase Polo-like kinase 2 (Plk2) was induced during prolonged epileptiform activity and was required for the activity-dependent reduction in membrane excitability of pyramidal neurons. Disruption of Plk2 function by dominant-negative or RNA interference not only blocked the downregulation of membrane excitability during epileptiform activity, but also unmasked a slow and progressive potentiation in synaptic strength that prevented the ability of the slice to undergo long-term potentiation. Thus, Plk2 function is required to prevent escalating potentiation and maintain synapses in a plastic state during epileptiform activity in hippocampal slice cultures.

Several studies have demonstrated a controversial involvement of NO in epileptogenesis. The aim of this study is to compare the NADPH diaphorase (NADPH-d) reactivity in the temporal cortex between surgical specimens of patients with intractable epilepsy and hippocampal sclerosis and autopsy controls. Brain samples of patients and postmortem controls were stained with the NADPH-d technique. Sprouting and larger areas of NADPH-d reactive neurons were found in the temporal cortex of epileptic patients.

To identify the roles of pyridoxal kinase (PLK) in epileptogenesis and the recovery mechanisms in spontaneous seizure, a chronological and comparative analysis of PLK expression in the gerbil hippocampus was conducted. PLK immunoreactivity in a pre-seizure group of seizure sensitive (SS) gerbils was more strongly detected than that in a seizure resistant (SR) group. The density of PLK immunoreactivity in a 30-min postictal group was significantly lower than that of a pre-seizure group. In a 12 h postictal group, PLK immunodensity recovered to pre-seizure level. The over-expression of PLK in the hippocampus of pre-seizure SS gerbils suggests that PLP play an important role in the modulation of GAD activity and GABA reuptake as mediated by membrane transporter via neurons.

Objectives: Previous studies have revealed an increase in several forms of phospholipase A2 activity associated with cell injury, but the secretory form of phospholipase A2 has not previously been studied in neurological disorders. We investigated the influence of seizures on secretory phospholipase A2 and phospholipid breakdown in synaptosome fractions prepared from rat hippocampus, cortex and cerebellum in pentylenetetrazol-induced epilepsy., Material and Methods: Secretory phospholipase A2 concentration was measured by a photometric enzyme immunoassay. The synaptosomes underwent extraction, and the phospholipids fractions for phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine were recovered from the thin layer chromatography plates. The amount of each phospholipid was quantified using the amount of recovered phosphate in each phospholipid spot., Results: Secretory phospholipase A2 concentration was found to be significantly higher in the epileptic group when compared with the control group. The amounts of phospholipids were found to be highly variable in different brain regions., Conclusion: Our results suggest that epileptic seizures enhanced phospholipid breakdown and induced alterations in the distribution of phospholipids in different brain regions.

The present studies demonstrate that expression of both type 5 and type 6 subunits of NADH dehydrogenase and the type 1 subunit of cytochrome oxidase is enhanced significantly in the brains of naive, epilepsy-prone EL mice. In contrast, no apparent change in expression occurred with type 1 and type 2 subunits of NADH dehydrogenase. When expression of type 5 and 6 subunits of NADH dehydrogenase was determined at 24 h after a single series of vestibular stimulation, significant down-regulation was detected. The expression of subunit 2 of NADH dehydrogenase augmented gradually after vestibular stimulation. The increased expression of these mitochondrial respiratory enzymes may reflect enhanced demand for energy due to inherent, spontaneous neuronal hyperactivity in the brains of EL mice.

The genetically epilepsy-prone rat (GEPR) is a model of generalized tonic/clonic epilepsy, and has functional noradrenergic deficiencies that act as partial determinants for the seizure predisposition and expression. The present study investigated the effect of repeated seizure experiences by acoustic stimulation (110 dB, 10 times) on the immunoreactivities of tyrosine hydroxylase (TH), a rate-determining enzyme in the synthesis of norepinephrine, in brain regions of GEPRs. TH immunoreactivity in locus coeruleus, the major noradrenergic nucleus in brain, was lower in GEPRs than control Sprague-Dawley rats. It was also decreased in several regions including inferior colliculus of GEPRs. Repeated experiences of audiogenic seizures further decreased TH immunoreactivities in locus coeruleus and inferior colliculus of GEPRs. The results from the present study suggest that the lower immunoreactivities of TH in locus coeruleus and inferior colliculus contribute, at least in part, to the noradrenergic deficits in GEPRs, and repeated seizure experiences further intensified these noradrenergic deficits, which may be related to the altered seizure expression by repetitive audiogenic seizure in GEPRs.

Several models that develop epileptiform discharges and epilepsy have been associated with a decrease in the activity of calmodulin-dependent kinase II. However, none of these studies has demonstrated a causal relationship between a decrease in calcium/calmodulin kinase II activity and the development of seizure activity. The present study was conducted to determine the effect of directly reducing calcium/calmodulin-dependent kinase activity on the development of epileptiform discharges in hippocampal neurons in culture. Complimentary oligonucleotides specific for the alpha subunit of the calcium/calmodulin kinase were used to decrease the expression of the enzyme. Reduction in kinase expression was confirmed by Western analysis, immunocytochemistry, and exogenous substrate phosphorylation. Increased neuronal excitability and frank epileptiform discharges were observed after a significant reduction in calmodulin kinase II expression. The epileptiform activity was a synchronous event and was not caused by random neuronal firing. Furthermore, the magnitude of decreased kinase expression correlated with the increased neuronal excitability. The data suggest that decreased calmodulin kinase II activity may play a role in epileptogenesis and the long-term plasticity changes associated with the development of pathological seizure activity and epilepsy.

Recent in vitro studies indicate an involvement of members of the interleukin-1beta converting enzyme (ICE) family of proteases in programmed neuronal cell death. Cell death of hippocampal neurons in animal models of cerebral ischemia and epilepsy shows morphological features of apoptosis and can be prevented by administration of protein synthesis inhibitors suggesting that de novo synthesis of components of the cell death program is necessary for neuronal apoptosis. In the present study we demonstrate by in situ hybridization analysis that expression of CPP-32, an ICE-related protease, is significantly upregulated in CA1 hippocampal neurons following global ischemia induced by cardiac arrest and in hippocampal neurons of the CA3/CA4 region after kainate-mediated epilepsy, respectively. Moreover, an increase in CPP-32-like proteolytic activity was detected in hippocampal extracts 24 h after ischemia using the fluorogenic CPP-32 substrate Ac-DEVD-AMC. Activation of CPP-32 clearly preceded cell death of hippocampal neurons as assessed by in situ end-labelling of nuclear DNA fragments. These results indicate that CPP-32 protease may be activated at both the transcriptional and post-translational level during neuronal apoptosis and that activation correlates with the selective vulnerability of hippocampal pyramidal neurons to ischemic and epileptic insults.

The regulation of neuronal nitric oxide synthase (NOS) mRNA levels during kindling epileptogenesis in the rat brain was investigated using in situ hybridization. Following 40 rapidly recurring seizures evoked by hippocampal stimulations, NOS mRNA expression decreased by 56% in the dentate granule cell layer (maximum at 2 h) and increased by 420,105 and 1260% in the CA1 and CA3 pyramidal layers and piriform cortex, respectively (maximum at 12-24 h). Gene expression had returned to control levels after one week. The presumed alterations of nitric oxide production, following the changes in NOS mRNA shown here, may modulate synaptic function during kindling development, and could influence neuronal vulnerability after epileptic insults.

The present study was designed to determine whether inhibitory neurons in human epileptic hippocampus are reduced in number, which could reduce inhibition on principal cells and thereby be a basis for seizure susceptibility. We studied the distribution of GABA neurons and puncta by using glutamate decarboxylase (GAD) immunocytochemistry (ICC) together with Nissl stains. Using quantitative comparisons of GAD-immunoreactive (GAD-IR) neurons and puncta in human epileptic hippocampus and in the normal monkey hippocampus, we found that GAD-IR neurons and puncta are relatively unaffected by the hippocampal sclerosis typical of hippocampal epilepsy where 50-90% of principal (non-GAD-IR) cells are lost. GAD-IR neurons and puncta were not significantly decreased compared with normal monkey. In 6 patients, prior in vivo electrophysiology demonstrated that the anterior hippocampus generated all seizures. The anterior and posterior hippocampus were processed simultaneously, and the counts of hippocampal GAD-IR neurons were numerically greater in anterior than in the posterior hippocampus, where no seizures were initiated. These results indicate that GABA neurons are intact in sclerotic and epileptogenic hippocampus. Computerized image analysis of puncta densities in fascia dentata, Ammon's horn, and subicular complex in epileptic hippocampi (n = 7) were not different from puncta densities in the same regions in normal monkey (n = 2). Hence, despite the significant loss of principal cells (50-90% loss) GABA terminals (GAD-IR puncta) were normal, which suggests GABA hyperinnervation of the remnant pyramidal cells and/or dendrites in human epileptic hippocampus. The apparent increase in puncta ranged from 2 (fascia dentata) to 3.3 (CA1) times normal puncta densities. These findings would suggest increased inhibition and less excitability; however, those regions were epileptogenic. We suggest that GABA terminal sprouting or hyperinnervation of the few remnant projection cells may serve to synchronize their membrane potentials so that subsequent excitatory inputs will trigger a larger population of neurons for seizure onset in the hippocampus and propagation out to undamaged regions of subiculum and neocortex.

The generation of focal cortical epilepsy as observed in human partial complex seizures is presumably due to enhanced physiologic responses or paroxysmal depolarization shifts (PDSs). However, the molecular mechanism that underlies these phenomena remains unknown. It could be due to a genetically determined error in a structural or regulatory protein or to posttranslational events that modulate membrane excitability. Since neither neuronal PDSs or interictal EEG spikes are sufficient to produce clinical epilepsy, the clinical expression of epilepsy may need the breakdown of neuronal or glial mechanisms that limit the spread of seizures. Hence, biochemical membrane studies of neurons and glia are necessary to understand the expression of human and experimental epilepsy. This chapter will review the role of glia in controlling neuronal excitability and neuron-glia relationships in experimental and human epilepsy. Data exploring the hypothesis that glial control of extracellular K+ or (K+)o is deficient in focal epilepsy induced by cold lesions will be reviewed. The role of glial carbonic anhydrase (CA) and glial control of putative amino acid transmitters in audiogenic epilepsy will be discussed. In the cold lesion, (K+)o activation constants of synaptosomal (Na+,K+)-ATPase are significantly decreased in the actively firing chronic focus, suggesting that the apparent affinity of the synaptosomal enzyme for K+ was increased within epileptic tissue that was actively firing. Interestingly, while sustained focal paroxysms could raise synaptosomal (Na+,K+)-ATPase, glial (Na+,K+)-ATPase and its activation by (K+)o remained decreased during sustained paroxysms in both acute and chronic lesions. Moreover, while the decrease of the absolute level of glial enzyme activity was less evident 45 days after lesion production, the poor response of glial enzyme to (K+)o never reversed to "normal" values. Hence, these experiments provided new information that glial (Na+,K+)-ATPase responds to K+ in a different manner when compared to synaptic enzyme. Glial ATPase and its activation by (K+)o remain decreased in either actively discharging acute lesions or in the indolent chronic foci. This could mean a reduction in the ability of glial membranes to maintain (K+)o homeostasis. As already suggested by Dichter, the impairment in glial control of elevated (K+)o could be mainly responsible for the transition of interictal discharges to ictal episodes, within the primary and the secondary foci.(ABSTRACT TRUNCATED AT 400 WORDS)

The activities of GABA-transaminase (GABA-T) were examined in several brain regions of amygdala-kindled rats, pretreated either with or without gabaculine, an irreversible GABA-T inhibitor. Histochemical and biochemical studies demonstrated that GABA-T activities decreased significantly in some brain regions 16 h after the gabaculine treatment. In contrast, no such alteration was detected in kindled animals after a 48-h survival period either with or without the pharmacological manipulation. The present results suggest that kindling causes retardation of GABA-T resynthesis in neurons, since the GABA-T activities detected 16 h after the drug treatment are due to newly synthetized enzyme in presumptive GABA neurons but not glial cells.