Showing papers on "Epileptogenesis published in 2011"

Critical review of current animal models of seizures and epilepsy used in the discovery and development of new antiepileptic drugs.

Abstract: Animal models for seizures and epilepsy have played a fundamental role in advancing our understanding of basic mechanisms underlying ictogenesis and epileptogenesis and have been instrumental in the discovery and preclinical development of novel antiepileptic drugs (AEDs). However, there is growing concern that the efficacy of drug treatment of epilepsy has not substantially improved with the introduction of new AEDs, which, at least in part, may be due to the fact that the same simple screening models, i.e., the maximal electroshock seizure (MES) and s.c. pentylenetetrazole (PTZ) seizure tests, have been used as gatekeepers in AED discovery for >6 decades. It has been argued that these old models may identify only drugs that share characteristics with existing drugs, and are unlikely to have an effect on refractory epilepsies. Indeed, accumulating evidence with several novel AEDs, including levetiracetan, has shown that the MES and PTZ models do not identify all potential AEDs but instead may fail to discover compounds that have great potential efficacy but work through mechanisms not tested by these models. Awareness of the limitations of acute seizure models comes at a critical crossroad. Clearly, preclinical strategies of AED discovery and development need a conceptual shift that is moving away from using models that identify therapies for the symptomatic treatment of epilepsy to those that may be useful for identifying therapies that are more effective in the refractory population and that may ultimately lead to an effective cure in susceptible individuals by interfering with the processes underlying epilepsy. To realize this goal, the molecular mechanisms of the next generation of therapies must necessarily evolve to include targets that contribute to epileptogenesis and pharmacoresistance in relevant epilepsy models.

Mechanisms of epileptogenesis and potential treatment targets.

Abstract: Prevention of epileptogenesis after brain trauma is an unmet medical challenge. Recent molecular profiling studies have provided an insight into molecular changes that contribute to formation of ictogenic neuronal networks, including genes regulating synaptic or neuronal plasticity, cell death, proliferation, and inflammatory or immune responses. These mechanisms have been targeted to prevent epileptogenesis in animal models. Favourable effects have been obtained using immunosuppressants, antibodies blocking adhesion of leucocytes to endothelial cells, gene therapy driving expression of neurotrophic factors, pharmacological neurostimulation, or even with conventional antiepileptic drugs by administering them before the appearance of genetic epilepsy. Further studies are needed to clarify the optimum time window and aetiological specificity of treatments. Questions related to adverse events also need further consideration. Encouragingly, the recent experimental studies emphasise that the complicated process of epileptogenesis can be favourably modified, and that antiepileptogenesis as a treatment indication might not be an impossible mission.

The ketogenic diet inhibits the mammalian target of rapamycin (mTOR) pathway

Abstract: The ketogenic diet (KD) is an effective treatment for epilepsy, but its mechanisms of action are poorly understood. We investigated the hypothesis that the KD inhibits mammalian target of rapamycin (mTOR) pathway signaling. The expression of pS6 and pAkt, markers of mTOR pathway activation, was reduced in hippocampus and liver of rats fed KD. In the kainate model of epilepsy, KD blocked the hippocampal pS6 elevation that occurs after status epilepticus. Because mTOR signaling has been implicated in epileptogenesis, these results suggest that the KD may have anticonvulsant or antiepileptogenic actions via mTOR pathway inhibition.

Rapamycin suppresses mossy fiber sprouting but not seizure frequency in a mouse model of temporal lobe epilepsy.

Abstract: Temporal lobe epilepsy is prevalent and can be difficult to treat effectively. Granule cell axon (mossy fiber) sprouting is a common neuropathological finding in patients with mesial temporal lobe epilepsy, but its role in epileptogenesis is unclear and controversial. Focally infused or systemic rapamycin inhibits the mammalian target of rapamycin (mTOR) signaling pathway and suppresses mossy fiber sprouting in rats. We tested whether long-term systemic treatment with rapamycin, beginning 1 d after pilocarpine-induced status epilepticus in mice, would suppress mossy fiber sprouting and affect the development of spontaneous seizures. Mice that had experienced status epilepticus and were treated for 2 months with rapamycin displayed significantly less mossy fiber sprouting (42% of vehicle-treated animals), and the effect was dose dependent. However, behavioral and video/EEG monitoring revealed that rapamycin- and vehicle-treated mice displayed spontaneous seizures at similar frequencies. These findings suggest mossy fiber sprouting is neither pro- nor anti-convulsant; however, there are caveats. Rapamycin treatment also reduced epilepsy-related hypertrophy of the dentate gyrus but did not significantly affect granule cell proliferation, hilar neuron loss, or generation of ectopic granule cells. These findings are consistent with the hypotheses that hilar neuron loss and ectopic granule cells might contribute to temporal lobe epileptogenesis.

Cytokines and epilepsy.

Abstract: Epilepsy is a common chronic neurological disorder affecting approximately 8 out of 1000 people. Its pathophysiology, however, has remained elusive in many regards. Consequently, adequate seizure control is still lacking in about one third of patients. Cytokines are soluble mediators of cell communication that are critical in immune regulation. In recent years, studies have shown that epileptic seizures can induce the production of cytokines, which in turn influence the pathogenesis and course of epilepsies. At the time of this review, the focus is mostly on interleukin-1beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNFα). In this review, we summarize the current knowledge regarding these cytokines and their potential roles in epilepsy. The focus concentrates on their expression and influence on induced seizures in animal models of epilepsy, as well as findings in human studies. Both proconvulsive and anticonvulsive effects have been reported for each of these molecules. One possible explanation for this phenomenon is that cytokines play dichotomous roles through multiple pathways, each of which is dependent on free concentration and available receptors. Furthermore, the immune-mediated leakage in the blood-brain-barrier also plays an important role in epileptogenesis. Nonetheless, these observations demonstrate the multifarious nature of cytokine networks and the complex relationship between the immune system and epilepsy. Future studies are warranted to further clarify the influence of the immune system on epilepsy and vice versa.

Brain Infiltration of Leukocytes Contributes to the Pathophysiology of Temporal Lobe Epilepsy

Abstract: Clinical and experimental evidence indicates that inflammatory processes contribute to the pathophysiology of epilepsy, but underlying mechanisms remain mostly unknown. Using immunohistochemistry for CD45 (common leukocyte antigen) and CD3 (T-lymphocytes), we show here microglial activation and infiltration of leukocytes in sclerotic tissue from patients with mesial temporal lobe epilepsy (TLE), as well as in a model of TLE (intrahippocampal kainic acid injection), characterized by spontaneous, nonconvulsive focal seizures. Using specific markers of lymphocytes, microglia, macrophages, and neutrophils in kainate-treated mice, we investigated with pharmacological and genetic approaches the contribution of innate and adaptive immunity to kainate-induced inflammation and neurodegeneration. Furthermore, we used EEG analysis in mutant mice lacking specific subsets of lymphocytes to explore the significance of inflammatory processes for epileptogenesis. Blood-brain barrier disruption and neurodegeneration in the kainate-lesioned hippocampus were accompanied by sustained ICAM-1 upregulation, microglial cell activation, and infiltration of CD3(+) T-cells. Moreover, macrophage infiltration was observed, selectively in the dentate gyrus where prominent granule cell dispersion was evident. Unexpectedly, depletion of peripheral macrophages by systemic clodronate liposome administration affected granule cell survival. Neurodegeneration was aggravated in kainate-lesioned mice lacking T- and B-cells (RAG1-knock-out), because of delayed invasion by Gr-1(+) neutrophils. Most strikingly, these mutant mice exhibited early onset of spontaneous recurrent seizures, suggesting a strong impact of immune-mediated responses on network excitability. Together, the concerted action of adaptive and innate immunity triggered locally by intrahippocampal kainate injection contributes seizure-suppressant and neuroprotective effects, shedding new light on neuroimmune interactions in temporal lobe epilepsy.

Brain inflammation as a biomarker in epilepsy.

Abstract: Experimental and clinical evidence have demonstrated the increased synthesis of specific inflammatory mediators, and the upregulation of their cognate receptors in the chronic epileptic brain, indicating that some proinflammatory pathways are activated in seizure foci. Inhibition of experimental seizures by pharmacological interference with specific proinflammatory signaling, together with evidence of changes in intrinsic susceptibility to seizures in transgenic mice with perturbed inflammatory pathways, was instrumental to establish the concept that brain inflammation has a role in the etiopathogenesis of seizures. Increasing evidence also highlights the possible involvement of inflammatory processes arising in the injured brain in the development of epilepsy (i.e., in epileptogenesis). Since brain inflammation in epilepsy is not a mere epiphenomenon of the pathology but is likely involved in the mechanisms underlying neuronal hyperexcitability, the onset of seizures and their recurrence, it might be considered as a biomarker of disease development and severity, and, as such, could be used for diagnostic, prognostic or therapeutic purposes, provided that adequate noninvasive methodologies are developed to detect and quantify brain inflammation in humans.

Molecular cascades that mediate the influence of inflammation on epilepsy.

Abstract: Experimental evidence strongly indicates a significant role for inflammatory and immune mediators in initiation of seizures and epileptogenesis. Here we will summarize data supporting the involvement of IL-1β, TNF-α and toll-like receptor 4 in seizure generation and the process of epileptogenesis. The physiological homeostasis and control over brain immune response depends on the integrity of the blood-brain barrier, transforming growth factor (TGF)-β signaling and leukocyte migration. To what extent targeting the immune system is successful in preventing epileptogenesis, and which signaling pathway should be beleaguered is still under intensive research.

Ablation of Cyclooxygenase-2 in Forebrain Neurons is Neuroprotective and Dampens Brain Inflammation after Status Epilepticus

Abstract: Cyclooxygenase-2 (COX-2), a source of inflammatory mediators and a multifunctional neuronal modulator, is rapidly induced in select populations of cortical neurons after status epilepticus. The consequences of rapid activity-triggered induction of COX-2 in neurons have been the subject of much study and speculation. To address this issue directly, we created a mouse in which COX-2 is conditionally ablated in selected forebrain neurons. Results following pilocarpine-induced status epilepticus indicate that neuronal COX-2 promotes early neuroprotection and then delayed neurodegeneration of CA1 pyramidal neurons, promotes neurodegeneration of nearby somatostatin interneurons in the CA1 stratum oriens and dentate hilus (which themselves do not express COX-2), intensifies a broad inflammatory reaction involving numerous cytokines and other inflammatory mediators in the hippocampus, and is essential for development of a leaky blood–brain barrier after seizures. These findings point to a profound role of seizure-induced neuronal COX-2 expression in neuropathologies that accompany epileptogenesis.

Ablation of Cyclooxygenase-2 in Forebrain Neurons is Neuroprotective and Dampens Brain Inflammation after Status Epilepticus

Abstract: Cyclooxygenase-2 (COX-2), a source of inflammatory mediators and a multifunctional neuronal modulator, is rapidly induced in select populations of cortical neurons after status epilepticus. The consequences of rapid activity-triggered induction of COX-2 in neurons have been the subject of much study and speculation. To address this issue directly, we created a mouse in which COX-2 is conditionally ablated in selected forebrain neurons. Results following pilocarpine-induced status epilepticus indicate that neuronal COX-2 promotes early neuroprotection and then delayed neurodegeneration of CA1 pyramidal neurons, promotes neurodegeneration of nearby somatostatin interneurons in the CA1 stratum oriens and dentate hilus (which themselves do not express COX-2), intensifies a broad inflammatory reaction involving numerous cytokines and other inflammatory mediators in the hippocampus, and is essential for development of a leaky blood–brain barrier after seizures. These findings point to a profound role of seizure-induced neuronal COX-2 expression in neuropathologies that accompany epileptogenesis.

Upregulation of adenosine kinase in astrocytes in experimental and human temporal lobe epilepsy.

Abstract: Purpose: Adenosine kinase (ADK) represents the key metabolic enzyme for the regulation of extracellular adenosine levels in the brain. In adult brain, ADK is primarily present in astrocytes. Several lines of experimental evidence support a critical role of ADK in different types of brain injury associated with astrogliosis, which is also a prominent morphologic feature of temporal lobe epilepsy (TLE). We hypothesized that dysregulation of ADK is an ubiquitous pathologic hallmark of TLE. Methods: Using immunocytochemistry and Western blot analysis, we investigated ADK protein expression in a rat model of TLE during epileptogenesis and the chronic epileptic phase and compared those findings with tissue resected from TLE patients with mesial temporal sclerosis (MTS). Key Findings: In rat control hippocampus and cortex, a low baseline expression of ADK was found with mainly nuclear localization. One week after the electrical induction of status epilepticus (SE), prominent up-regulation of ADK became evident in astrocytes with a characteristic cytoplasmic localization. This increase in ADK persisted at least for 3-4 months after SE in rats developing a progressive form of epilepsy. In line with the findings from the rat model, expression of astrocytic ADK was also found to be increased in the hippocampus and temporal cortex of patients with TLE. In addition, in vitro experiments in human astrocyte cultures showed that ADK expression was increased by several proinflammatory molecules (interleukin-1β and lipopolysaccharide). Significance: These results suggest that dysregulation of ADK in astrocytes is a common pathologic hallmark of TLE. Moreover, in vitro data suggest the existence of an additional layer of modulatory crosstalk between the astrocyte-based adenosine cycle and inflammation. Whether this interaction also can play a role in vivo needs to be further investigated.

Inflammatory changes during epileptogenesis and spontaneous seizures in a mouse model of mesiotemporal lobe epilepsy.

Abstract: Summary Purpose: Neuroinflammation appears as a prominent feature of the mesiotemporal lobe epilepsy syndrome (MTLE) that is observed in human patients and animal models. However, the precise temporal relationship of its development during epileptogenesis remains to be determined. The aim of the present study was to investigate (1) the time course and spatial distribution of neuronal death associated with seizure development, (2) the time course of microglia and astrocyte activation, and (3) the kinetics of induction of mRNAs from neuroinflammatory-related proteins during the emergence of recurrent seizures. Methods: Experimental MTLE was induced by the unilateral intrahippocampal injection of kainate in C57BL/6 adult mice. Microglial and astrocytic changes in both ipsilateral and contralateral hippocampi were examined by respectively analyzing griffonia simplicifolia (GSA) lectin staining and glial fibrillary acidic protein (GFAP) immunoreactivity. Changes in mRNA levels of selected genes of cytokine and cytokine regulatory proteins (interleukin-1β, IL-1β; interleukin-1 receptor antagonist, IL-1Ra; suppressor of cytokine signaling 3, SOCS3) and enzymes of the eicosanoid pathway (group IVA cytosolic phospholipase A2, cPLA2-α; cycloxygenase-2, COX-2) were studied by reverse transcription-quantitative real time polymerase chain reaction. Key Findings: Our data show an immediate cell death occurring in the kainate-injected hippocampus during the initial status epilepticus (SE). A rapid increase of activated lectin-positive cells and GFAP-immunoreactivity was subsequently detected in the ipsilateral hippocampus. In the same structure, Il-1β, IL-1Ra, and COX-2 mRNA were specifically increased during SE and epileptogenesis with a different time course. Conversely, the expression of SOCS3 mRNA, a surrogate marker of interleukin signaling, was mainly increased in the contralateral hippocampus after SE. Significance: Our data show that specific neuroinflammatory pathways are activated in a time- and structure-dependent manner with putative distinct roles in epileptogenesis.

Blood-brain barrier dysfunction, status epilepticus, seizures and epilepsy: a puzzle of a chicken and egg?

Abstract: Status epilepticus is often associated with endothelial dysfunction and increased vessels permeability. The direct role of blood-brain barrier (BBB) dysfunction in epileptogenesis and brain damage is discussed in the paragraphs below. On the cellular level, astrocytes are the early responders to the efflux of serum proteins in the presence of dysfunctional BBB. Astrocytic responses include the activation of the innate immune system and disturbed homeostasis of extracellular potassium and glutamate. These astrocytic changes, in turn, are associated with enhanced excitability of neurons and altered network connectivity. Transforming growth factor beta (TGF-β) signaling appears to be a critical pathway in the astrocytic response to serum albumin and thus may be a potential new target for the prevention of epileptogenesis and secondary damage following status epilepticus. Prolonged seizure and status epilepticus (SE) are neurological emergencies which may be followed by the development of unprovoked seizures as well as mental and neurologic deficits. Under physiological conditions, seizures are associated with a robust vascular response (vasodilation) and increased regional cerebral blood flow. While this neurovascular coupling may be considered as a physiological homeostatic response to increased metabolic demand, recent animal and human data suggest that under pathological conditions the physiological coupling may fail, and neuronal depolarization may be associated with no or “inverse coupling” – i.e. vasoconstriction (Dreier, 2011). Pathological vascular response may lead to reduced energy supply and worsening of the tissue metabolic state, thus promoting cellular damage and slowing energy-demanding homeostatic mechanisms such as active transporters required for neuronal repolarization. These changes will prolong neuronal depolarization and delay the termination of seizures. In addition, a metabolic compromise may also be associated with functional changes within vascular endothelial cells, leading to increased vascular permeability. Indeed, vascular dysfunction and increased permeability of the blood-brain barrier (BBB) have been documented following SE as well as under different common brain insults. The “chicken and egg” dilemma directly questions the role of BBB dysfunction in the pathophysiology of brain damage associated with SE. In this presentation I will discuss the direct role of BBB dysfunction in SE, epileptogenesis and brain damage. The blood-brain barrier (BBB) is a functional and structural complex barrier characterizing the vasculature within the central nervous system and is crucial for the maintenance of strict extracellular environment. Recent studies in pilocarpine-exposed rats (van Vliet et al., 2007) described increased number of spontaneous seizures in animals showing greater BBB dysfunction following SE, suggesting a potential direct role for BBB dysfunction in epileptogenesis. Indeed, our experiments in rodents demonstrated that dysfunction of the BBB underlies the initiation of transcriptional program within the neurovascular network (Cacheaux et al., 2009). This rapid transcriptional response is associated within few hours with significant changes in the function of astrocytes and microglia, and includes upregulation of cytokines and chemokines. In-vitro experiments in the acute slice preparation show that the functional transformation of astrocytes is specifically associated with disturbed extracellular homeostasis leading to activity-dependent accumulation of potassium and glutamate in the extracellular space (David et al., 2009). These are followed by neuronal depolarization, slower spike repolarization, increased transmitter release, enhancement of glutamate content in the synaptic cleft as well as activation of NMDA receptors and calcium influx. In turn, short-term synaptic facilitation and long-term synaptic modifications occur. The potential outcome of these changes was observed using in-vivo recording showing that BBB opening was sufficient to result in the development of spontaneous unprovoked seizures 4-10 days after treatment in >80% of the animals. Sensory-motor neurological dysfunction developed 3-5 weeks after focal BBB opening in the corresponding region of the neocortex, and was associated with loss of cortical volume (measured using in-vivo MRI imaging), reduced dendritic branching and neuronal loss with lasting astrogliosis (Tomkins et al., 2007). The mechanisms underlying epileptogenesis and neuronal damage in the presence of BBB dysfunction are only partly understood. Specific attention has been given to serum albumin, which diffuses into the neuropil in SE-exposed animals and is transported into different populations of cells, probably via different mechanisms. Interestingly, while hours following the initiation of SE a selective uptake into astroglial cell populations has been found, 1-2 days later serum albumin (and IgG) are found within principle hippocampal neurons. Direct exposure of brain tissue to albumin was associated with astroglial response via transforming growth factor beta (TGF-β) signaling and phosphorylation of the Smad-2/5 pathway (Ivens et al., 2007;Cacheaux et al., 2009). Experimental data further suggest that blocking TGF-β signaling following experimental BBB opening, decreases albumin-induced transcriptional response and prevents epileptogenesis. Finally, although limited, clinical data support a frequent BBB dysfunction in human patients with post traumatic epilepsy (Tomkins et al., 2008) and its promoting effect in the development of seizures in patients with tumors (Marchi et al., 2007). These studies point to the critical role of pathological neurovascular interactions in astroglial dysfunction, immune response, neuronal hyperexcitability and delayed network dysfunction and degeneration in the SE-exposed brain. Future studies are awaited to better diagnose vascular functions in clinical settings, explore their use as biomarkers for outcome and choice of treatment, and their potential as targets for treatment (Friedman et al., 2009). The future development of new biomarkers and imaging approaches for the diagnosis of vascular and immune functions may be critical to allow specific treatments that will be tailored to the principle pathophysiological mechanism(s) in an individual patient during and following status epilepticus.

The methylation hypothesis: Do epigenetic chromatin modifications play a role in epileptogenesis?

Abstract: Any structural brain lesion can provoke epilepsy, although onset and progression of seizures as well as response to antiepileptic drug (AED) treatment remain difficult to predict in each patient. Tremendous work has focused on the development of new AED compounds with the intention to treat seizures. However, these efforts have not yet discovered a "magic bullet" that cures epilepsy in every patient or modifies disease progression. With the "methylation hypothesis" we propose that epigenetic mechanisms play a pivotal role in epileptogenesis in patients with structural lesions. "Epigenetics" is defined as information that is heritable during cell division other than the DNA sequence itself, that is, DNA methylation or histone tail modifications, which can produce lasting alterations in chromatin structure and gene expression. They are increasingly recognized as fundamental regulatory processes in central nervous system development, synaptic plasticity, and memory, and also play a role in neurologic disorders such as schizophrenia and spinal muscular atrophy. The methylation hypothesis suggests that seizures by themselves can induce epigenetic chromatin modifications, thereby aggravating the epileptogenic condition. The impact of the methylation hypothesis for new-onset epilepsy will be discussed. Unravelling of epigenetic pathomechanisms will also open new strategies to identify molecular targets for pharmacologic treatment in epilepsies.

Anomalous levels of Cl(-) transporters cause a decrease of GABAergic inhibition in human peritumoral epileptic cortex

Abstract: Purpose Several factors contribute to epileptogenesis in patients with brain tumors, including reduced γ-aminobutyric acid (GABA)ergic inhibition. In particular, changes in Cl(-) homeostasis in peritumoral microenvironment, together with alterations of metabolism, are key processes leading to epileptogenesis in patients afflicted by glioma. It has been recently proposed that alterations of Cl(-) homeostasis could be involved in tumor cell migration and metastasis formation. In neurons, the regulation of intracellular Cl(-) concentration ([Cl(-) ](i) ) is mediated by NKCC1 and KCC2 transporters: NKCC1 increases while KCC2 decreases [Cl(-) ](i) . Experiments were thus designed to investigate whether, in human epileptic peritumoral cortex, alterations in the balance of NKCC1 and KCC2 activity may decrease the hyperpolarizing effects of GABA, thereby contributing to epileptogenesis in human brain tumors. Methods Membranes from peritumoral cortical tissues of epileptic patients afflicted by gliomas (from II to IV WHO grade) and from cortical tissues of nonepileptic patients were injected into Xenopus oocytes leading to the incorporation of functional GABA(A) receptors. The GABA-evoked currents were recorded using standard two-microelectrode voltage-clamp technique. In addition, immunoblot analysis and immunohistochemical staining were carried out on membranes and tissues from the same patients. Key findings We found that in oocytes injected with epileptic peritumoral cerebral cortex, the GABA-evoked currents had a more depolarized reversal potential (E(GABA) ) compared to those from nonepileptic healthy cortex. This difference of E(GABA) was abolished by the NKCC1 blocker bumetanide or unblocking of KCC2 with the Zn(2+) chelator TPEN. Moreover, Western blot analysis revealed an increased expression of NKCC1, and more modestly, of KCC2 transporters in epileptic peritumoral tissues compared to nonepileptic control tissues. In addition, NKCC1 immunoreactivity was strongly increased in peritumoral cortex with respect to nonepileptic cortex, with a prominent expression in neuronal cells. Significance We report that the positive shift of E(GABA) in epileptic peritumoral human cortex is due to an altered expression of NKCC1 and KCC2, perturbing Cl(-) homeostasis, which might lead to a consequent reduction in GABAergic inhibition. These findings point to a key role of Cl(-) transporters KCC2 and NKCC1 in tumor-related epilepsy, suggesting a more specific drug therapy and surgical approaches for the epileptic patients afflicted by brain tumors.

Recessive loss-of-function mutation in the pacemaker HCN2 channel causing increased neuronal excitability in a patient with idiopathic generalized epilepsy

Abstract: The hyperpolarization-activated I h current, coded for by hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, controls synaptic integration and intrinsic excitability in many brain areas. Because of their role in pacemaker function, defective HCN channels are natural candidates for contributing to epileptogenesis. Indeed, I h is pathologically altered after experimentally induced seizures, and several independent data indicate a link between dysfunctional HCN channels and different forms of epilepsy. However, direct evidence for functional changes of defective HCN channels correlating with the disease in human patients is still elusive. By screening families with epilepsy for mutations in Hcn1 and Hcn2 genes, we found a recessive loss-of-function point mutation in the gene coding for the HCN2 channel in a patient with sporadic idiopathic generalized epilepsy. Of 17 screened members of the same family, the proband was the only one affected and homozygous for the mutation. The mutation (E515K) is located in the C-linker, a region known to affect channel gating. Functional analysis revealed that homomeric mutant, but not heteromeric wild-type/mutant channels, have a strongly inhibited function caused by a large negative shift of activation range and slowed activation kinetics, effectively abolishing the HCN2 contribution to activity. After transfection into acutely isolated newborn rat cortical neurons, homomeric mutant, but not heteromeric wild type/mutant channels, lowered the threshold of action potential firing and strongly increased cell excitability and firing frequency when compared with wild-type channels. This is the first evidence in humans for a single-point, homozygous loss-of-function mutation in HCN2 potentially associated with generalized epilepsy with recessive inheritance.

Targeting BK (big potassium) Channels in Epilepsy

Abstract: Introduction: Epilepsies are disorders of neuronal excitability characterized by spontaneous and recurrent seizures. Ion channels are critical for regulating neuronal excitability and, therefore, can contribute significantly to epilepsy pathophysiology. In particular, large conductance, Ca2+-activated K+ (BKCa) channels play an important role in seizure etiology. These channels are activated by both membrane depolarization and increased intracellular Ca2+. This unique coupling of Ca2+ signaling to membrane depolarization is important in controlling neuronal hyperexcitability, as outward K+ current through BKCa channels hyperpolarizes neurons. Areas covered: BKCa channel structure–function and the role of these channels in epilepsy pathophysiology. Expert opinion: Loss-of-function BKCa channel mutations contribute to neuronal hyperexcitability that can lead to temporal lobe epilepsy, tonic–clonic seizures and alcohol withdrawal seizures. Similarly, BKCa channel blockade can trigger seizures and status epil...

Role of Anticonvulsant and Antiepileptogenic Neurosteroids in the Pathophysiology and Treatment of Epilepsy

Abstract: This review highlights the role of major endogenous neurosteroids in seizure disorders and the promise of neurosteroid replacement therapy in epilepsy. Neurosteroids are endogenous modulators of seizure susceptibility. Neurosteroids such as allopregnanolone (3a-hydroxy-5a-pregnane-20-one) and allotetrahydrodeoxycorticosterone (3a,21-dihydroxy-5a-pregnan-20-one) are positive modulators of GABA-A receptors. Aside from peripheral tissues, neurosteroids are synthesized within the brain, mostly in principal neurons. Neurosteroids potentiate synaptic GABA-A receptor function and also activate delta-subunit-containing extrasynaptic GABA-A receptors that mediate tonic currents and thus may play an important role in neuronal network excitability and seizure susceptibility. Our studies over the past decade have shown that neurosteroids are broad-spectrum anticonvulsants and confer seizure protection in various animal models. They protect against seizures induced by GABA-A receptor antagonists, 6-Hz model, pilocarpine-induced limbic seizures and seizures in kindled animals. Unlike benzodiazepines, tolerance does not occur to their actions during chronic administration. Our recent studies provide compelling evidence that neurosteroids may have antiepileptogenic properties. There is emerging evidence that endogenous neurosteroids may play a key role in the pathophysiology of catamenial epilepsy, stress-sensitive seizure conditions, temporal lobe epilepsy, and alcohol-withdrawal seizures. It is suggested that neurosteroid replacement with natural or synthetic neurosteroids may be useful in the treatment of epilepsy. Synthetic analogs of neurosteroids that are devoid of hormonal side effects show promise in the treatment of diverse seizure disorders. Agents that stimulate endogenous production of neurosteroids may also be useful for treatment of epilepsy.

In vivo activation of endocannabinoid system in temporal lobe epilepsy with hippocampal sclerosis

Abstract: The endocannabinoid system modulates neuronal excitability, protects neurons against hyperexcitability and is involved in epileptogenesis in animal models of mesial temporal lobe epilepsy with hippocampal sclerosis. We performed in vivo positron emission tomography imaging of the type 1 cannabinoid receptor in patients with mesial temporal lobe epilepsy with hippocampal sclerosis. Twelve patients with refractory mesial temporal lobe epilepsy due to hippocampal sclerosis received a [18F]MK-9470 scan to assess type 1 cannabinoid receptor availability in vivo . Parametric modified standard uptake values were used as quantitative measure of type 1 cannabinoid receptor availability and images were spatially normalized to standard space. Voxel-based analysis was performed comparing patients with hippocampal sclerosis to controls and correlations between type 1 cannabinoid receptor status and seizure characteristics were done using volumes of interest. Type 1 cannabinoid receptor positron emission tomography was co-registered with subtraction ictal single photon emission computed tomography co-registered to magnetic resonance imaging of a complex partial seizure ( n = 9). An increased type 1 cannabinoid receptor availability in the ipsilateral temporal lobe was observed, which correlated negatively with the latency since last seizure before scanning and positively to the number of seizures in the month before scanning. A decreased type 1 cannabinoid receptor availability was present in the superior insular cortex, ipsilateral more than contralateral. The ipsilateral insular region displayed a mild ictal hyperperfusion in the transition zone of subtraction ictal single photon emission computed tomography co-registered to magnetic resonance imaging temporal lobe hyperperfusion-frontal lobe hypoperfusion during complex partial seizures. Type 1 cannabinoid receptor availability showed opposite changes in different brain regions that are involved during complex partial seizures in refractory mesial temporal lobe epilepsy with hippocampal sclerosis. The increase in type 1 cannabinoid receptor availability at the seizure onset zone might be a protective mechanism of neurons against hyperexcitability and seizure activity, or contribute to the process of epileptogenesis, or both. The decreased type 1 cannabinoid receptor availability in the insula may play a role in surround inhibition and prevention of seizure propagation. * Abbreviations : CB1R : type 1 cannabinoid receptor SISCOM : subtraction ictal single photon emission computed tomography co-registered to MRI

Altered GABA Signaling in Early Life Epilepsies

Abstract: The incidence of seizures is particularly high in the early ages of life. The immaturity of inhibitory systems, such as GABA, during normal brain development and its further dysregulation under pathological conditions that predispose to seizures have been speculated to play a major role in facilitating seizures. Seizures can further impair or disrupt GABAA signaling by reshuffling the subunit composition of its receptors or causing aberrant reappearance of depolarizing or hyperpolarizing GABAA receptor currents. Such effects may not result in epileptogenesis as frequently as they do in adults. Given the central role of GABAA signaling in brain function and development, perturbation of its physiological role may interfere with neuronal morphology, differentiation, and connectivity, manifesting as cognitive or neurodevelopmental deficits. The current GABAergic antiepileptic drugs, while often effective for adults, are not always capable of stopping seizures and preventing their sequelae in neonates. Recent studies have explored the therapeutic potential of chloride cotransporter inhibitors, such as bumetanide, as adjunctive therapies of neonatal seizures. However, more needs to be known so as to develop therapies capable of stopping seizures while preserving the age- and sex-appropriate development of the brain.

Early life stress enhancement of limbic epileptogenesis in adult rats: mechanistic insights.

Abstract: Background Exposure to early postnatal stress is known to hasten the progression of kindling epileptogenesis in adult rats. Despite the significance of this for understanding mesial temporal lobe epilepsy (MTLE) and its associated psychopathology, research findings regarding underlying mechanisms are sparse. Of several possibilities, one important candidate mechanism is early life ‘programming’ of the hypothalamic-pituitary-adrenal (HPA) axis by postnatal stress. Elevated corticosterone (CORT) in turn has consequences for neurogenesis and cell death relevant to epileptogenesis. Here we tested the hypotheses that MS would augment seizure-related corticosterone (CORT) release and enhance neuroplastic changes in the hippocampus. Methodology/Principal Findings Eight-week old Wistar rats, previously exposed on postnatal days 2–14 to either maternal separation stress (MS) or control brief early handling (EH), underwent rapid amygdala kindling. We measured seizure-induced serum CORT levels and post-kindling neurogenesis (using BrdU). Three weeks post-kindling, rats were euthanized for histology of the hippocampal CA3c region (pyramidal cell counts) and dentate gyrus (DG) (to count BrdU-labelled cells and measure mossy fibre sprouting). As in our previous studies, rats exposed to MS had accelerated kindling rates in adulthood. Female MS rats had heightened CORT responses during and after kindling (p<0.05), with a similar trend in males. In both sexes total CA3c pyramidal cell numbers were reduced in MS vs. EH rats post-kindling (p = 0.002). Dentate granule cell neurogenesis in female rats was significantly increased post-kindling in MS vs. EH rats. Conclusions/Significance These data demonstrate that early life stress results in enduring enhancement of HPA axis responses to limbic seizures, with increased hippocampal CA3c cell loss and augmented neurogenesis, in a sex-dependent pattern. This implicates important candidate mechanisms through which early life stress may promote vulnerability to limbic epileptogenesis in rats as well as to human MTLE and its associated psychiatric disorders.