Onset Of Spontaneous Seizures Research Articles

Combined value of interictal markers and stimulated seizures to estimate the seizure onset zone in stereoelectroencephalography.

This study was undertaken to investigate the potential of interictal electroencephalographic (EEG) findings and electrically stimulated seizures during stereo-EEG (SEEG) as surrogate markers for the spontaneous seizure onset zone (spSOZ). We hypothesized that combining the localizing information of these markers would allow clinically meaningful estimation of the spSOZ. We included all patients (n = 63) who underwent SEEG between January 2013 and March 2020 at Helsinki University Hospital and had spontaneous seizures during the recording. We scored spikes, gamma activity, and background abnormality on each channel visually during a 12-h epoch containing waking state and sleep. Based on semiology, we classified stimulated seizures as typical or atypical/unclassifiable and estimated the stimulated SOZ (stimSOZ) for typical seizures. To assess which markers increased the odds of channel inclusion in the spSOZ, we fitted mixed effects logistic regression models. A combined regression model including the stimSOZ and interictal markers scored during sleep performed better in estimating which channels were part of the spSOZ than models based on stimSOZ (p < .001) or interictal markers (p < .001) alone. Of the individual markers, the effect sizes were greatest for inclusion of a channel in the stimSOZ (odds ratio [OR] = 60, 95% confidence interval [CI] = 37-97, p < .001) and for continuous (OR = 25, 95% CI = 12-55, p < .001) and subcontinuous (OR = 36, 95% CI = 21-64, p < .001) interictal spiking. At the individual level, the model's accuracy to predict spSOZ inclusion varied markedly (median accuracy = 85.7, range = 54.4-100), which was not explained by etiology (p > .05). Compared to either marker alone, combining visually rated interictal SEEG markers and stimulated seizures improved prediction of which SEEG channels belonged to the spSOZ. Inclusion in the stimSOZ and continuous or subcontinuous spikes increased the odds of spSOZ inclusion the most. Future studies should investigate whether suboptimal sampling of the true epileptogenic zone can explain the model's poor performance in certain patients.

Disease modification upon brief exposure to tofacitinib during chronic epilepsy.

All current drug treatments for epilepsy, a neurological disorder affecting over 50 million people 1,2 merely treat symptoms, and a third of patients do not respond to medication. There are no disease modifying treatments that may be administered briefly to patients to enduringly eliminate spontaneous seizures and reverse cognitive deficits 3,4 . Applying network and systems-based approaches to rodent models and human temporal lobectomy samples, we observe the well-characterized pattern of rapid induction and subsequent quenching exhibited by the JAK/STAT pathway within days of epileptic insult. This is followed by an utterly unexpected, resurgent activation months later with the onset of spontaneous seizures. Targeting the first wave of activation after epileptic insult does not prevent disease. However, brief inhibition of the second wave with CP690550 (Tofacitinib) 5,6 enduringly suppresses seizures, rescues deficits in spatial memory, and restores neuropathological alterations to naïve levels. Seizure suppression lasts for at least 2 months after last dose. Using discovery-based transcriptomic analysis across models of epilepsy and validation of putative mechanisms with human data, we demonstrate a powerful approach to identifying disease modifying targets; this may be useful for other neurodegenerative diseases. With this approach, we find that reignition of inflammatory JAK/STAT3 signaling in chronic epilepsy opens a powerful window for disease modification with the FDA-approved, orally available drug CP690550.

Understanding epileptogenesis from molecules to network alteration.

Epilepsy is characterized by recurrent seizures. Following an initial insult, a latent period precedes the onset of spontaneous seizures, a process referred to as epileptogenesis. This period plays a critical role in halting the progression toward epilepsy before the onset of abnormal molecular and network alterations. In this study, the fundamental concepts of epileptogenesis as well as the associated molecular and cellular targets are reviewed.

Inhibition of Neuron-Restrictive Silencing Factor (REST/NRSF) Chromatin Binding Attenuates Epileptogenesis.

The mechanisms by which brain insults lead to subsequent epilepsy remain unclear. Insults including trauma, stroke, infections, and long seizures (status epilepticus, SE) increase the nuclear expression and chromatin binding of the neuron-restrictive silencing factor/RE-1 silencing transcription factor (NRSF/REST). REST/NRSF orchestrates major disruption of the expression of key neuronal genes, including ion channels and neurotransmitter receptors, potentially contributing to epileptogenesis. Accordingly, transient interference with REST/NRSF chromatin binding after an epilepsy-provoking SE suppressed spontaneous seizures for the 12 d duration of a prior study. However, whether the onset of epileptogenesis was suppressed or only delayed has remained unresolved. The current experiments determined if transient interference with REST/NRSF chromatin binding prevented epileptogenesis enduringly or, alternatively, slowed epilepsy onset. Epileptogenesis was elicited in adult male rats via systemic kainic acid-induced SE (KA-SE). We then determined if decoy, NRSF-binding-motif oligodeoxynucleotides (NRSE-ODNs), given twice following KA-SE (1) prevented REST/NRSF binding to chromatin, using chromatin immunoprecipitation, or (2) prevented the onset of spontaneous seizures, measured with chronic digital video-electroencephalogram. Blocking NRSF function transiently after KA-SE significantly lengthened the latent period to a first spontaneous seizure. Whereas this intervention did not influence the duration and severity of spontaneous seizures, total seizure number and seizure burden were lower in the NRSE-ODN compared with scrambled-ODN cohorts. Transient interference with REST/NRSF function after KA-SE delays and moderately attenuates insult-related hippocampal epilepsy, but does not abolish it. Thus, the anticonvulsant and antiepileptogenic actions of NRSF are but one of the multifactorial mechanisms generating epilepsy in the adult brain.

Systematic 1 Hz direct electrical stimulation for seizure induction: A reliable method for localizing seizure onset zone and predicting seizure freedom

ObjectiveTo prospectively investigate the utility of seizure induction using systematic 1 Hz stimulation by exploring its concordance with the spontaneous seizure onset zone (SOZ) and relation to surgical outcome; comparison with seizures induced by non-systematic 50 Hz stimulation was attempted as well. MethodsProspective cohort study from 2018 to 2021 with ≥ 1 y post-surgery follow up at Yale New Haven Hospital. With 1 Hz, all or most of the gray matter contacts were stimulated at 1, 5, and 10 mA for 30–60s. With 50 Hz, selected gray matter contacts outside of the medial temporal regions were stimulated at 1–5 mA for 0.5–3s. Stimulation was bipolar, biphasic with 0.3 ms pulse width. The Yale Brain Atlas was used for data visualization. Variables were analyzed using Fisher's exact, χ2, or Mann–Whitney test. ResultsForty-one consecutive patients with refractory epilepsy undergoing intracranial EEG for localization of SOZ were included. Fifty-six percent (23/41) of patients undergoing 1 Hz stimulation had seizures induced, 83% (19/23) habitual (clinically and electrographically). Eighty two percent (23/28) of patients undergoing 50 Hz stimulation had seizures, 65% (15/23) habitual. Stimulation of medial temporal or insular regions with 1 Hz was more likely to induce seizures compared to other regions [15/32 (47%) vs. 2/41 (5%), p < 0.001]. Sixteen patients underwent resection; 11/16 were seizure free at one year and all 11 had habitual seizures induced by 1 Hz; 5/16 were not seizure free at one year and none of those 5 had seizures with 1 Hz (11/11 vs 0/5, p < 0.0001). No patients had convulsions with 1 Hz stimulation, but four did with 50 Hz (0/41 vs. 4/28, p = 0.02). SignificanceInduction of habitual seizures with 1 Hz stimulation can reliably identify the SOZ, correlates with excellent surgical outcome if that area is resected, and may be superior (and safer) than 50 Hz for this purpose. However, seizure induction with 1 Hz was infrequent outside of the medial temporal and insular regions in this study.

Optogenetic activation of the superior colliculus attenuates spontaneous seizures in the pilocarpine model of temporal lobe epilepsy.

Decades of studies have indicated that activation of the deep and intermediate layers of the superior colliculus can suppress seizures in a wide range of experimental models of epilepsy. However, prior studies have not examined efficacy against spontaneous limbic seizures. The present study aimed to address this gap through chronic optogenetic activation of the superior colliculus in the pilocarpine model of temporal lobe epilepsy. Sprague Dawley rats underwent pilocarpine-induced status epilepticus and were maintained until the onset of spontaneous seizures. Virus coding for channelrhodopsin-2 was injected into the deep and intermediate layers of the superior colliculus, and animals were implanted with head-mounted light-emitting diodes at the same site. Rats were stimulated with either 5- or 100-Hz light delivery. Seizure number, seizure duration, 24-h seizure burden, and behavioral seizure severity were monitored. Both 5- and 100-Hz optogenetic stimulation of the deep and intermediate layers of the superior colliculus reduced daily seizure number and total seizure burden in all animals in the active vector group. Stimulation did not affect either seizure duration or behavioral seizure severity. Stimulation was without effect in opsin-negative control animals. Activation of the deep and intermediate layers of the superior colliculus reduces both the number of seizures and total daily seizure burden in the pilocarpine model of temporal lobe epilepsy. These novel data demonstrating an effect against chronic experimental seizures complement a long history of studies documenting the antiseizure efficacy of superior colliculus activation in a range of acute seizure models.

Post-Traumatic Epilepsy and Comorbidities: Advanced Models, Molecular Mechanisms, Biomarkers, and Novel Therapeutic Interventions

Post-traumatic epilepsy (PTE) is one of the most devastating long-term, network consequences of traumatic brain injury (TBI). There is currently no approved treatment that can prevent onset of spontaneous seizures associated with brain injury, and many cases of PTE are refractory to antiseizure medications. Post-traumatic epileptogenesis is an enduring process by which a normal brain exhibits hypersynchronous excitability after a head injury incident. Understanding the neural networks and molecular pathologies involved in epileptogenesis are key to preventing its development or modifying disease progression. In this article, we describe a critical appraisal of the current state of PTE research with an emphasis on experimental models, molecular mechanisms of post-traumatic epileptogenesis, potential biomarkers, and the burden of PTE-associated comorbidities. The goal of epilepsy research is to identify new therapeutic strategies that can prevent PTE development or interrupt the epileptogenic process and relieve associated neuropsychiatric comorbidities. Therefore, we also describe current preclinical and clinical data on the treatment of PTE sequelae. Differences in injury patterns, latency period, and biomarkers are outlined in the context of animal model validation, pathophysiology, seizure frequency, and behavior. Improving TBI recovery and preventing seizure onset are complex and challenging tasks; however, much progress has been made within this decade demonstrating disease modifying, anti-inflammatory, and neuroprotective strategies, suggesting this goal is pragmatic. Our understanding of PTE is continuously evolving, and improved preclinical models allow for accelerated testing of critically needed novel therapeutic interventions in military and civilian persons at high risk for PTE and its devastating comorbidities.Significance StatementPost-traumatic epilepsy is a chronic seizure condition after brain injury. With few models and limited understanding of the underlying progression of epileptogenesis, progress is extremely slow to find a preventative treatment for PTE. This study reviews the current state of modeling, pathology, biomarkers, and potential interventions for PTE and comorbidities. There’s new optimism in finding a drug therapy for preventing PTE in people at risk, such as after traumatic brain injury, concussion, and serious brain injuries, especially in military persons.

Chronic partial TrkB activation reduces seizures and mortality in a mouse model of Dravet syndrome

Dravet syndrome (DS) is one of the most severe childhood epilepsies, characterized by intractable seizures and comorbidities including cognitive and social dysfunction and high premature mortality. DS is mainly caused by loss-of-function mutations in the Scn1a gene encoding Nav1.1 that is predominantly expressed in inhibitory parvalbumin-containing (PV) interneurons. Decreased Nav1.1 impairs PV cell function, contributing to DS phenotypes. Effective pharmacological therapy that targets defective PV interneurons is not available. The known role of brain-derived neurotrophic factor (BDNF) in the development and maintenance of interneurons, together with our previous results showing improved PV interneuronal function and antiepileptogenic effects of a TrkB receptor agonist in a posttraumatic epilepsy model, led to the hypothesis that early treatment with a TrkB receptor agonist might prevent or reduce seizure activity in DS mice. To test this hypothesis, we treated DS mice with LM22A-4 (LM), a partial agonist at the BDNF TrkB receptor, for 7 d starting at postnatal day 13 (P13), before the onset of spontaneous seizures. Results from immunohistochemistry, Western blot, whole-cell patch-clamp recording, and invivo seizure monitoring showed that LM treatment increased the number of perisomatic PV interneuronal synapses around cortical pyramidal cells in layer V, upregulated Nav1.1 in PV neurons, increased inhibitory synaptic transmission, and decreased seizures and the mortality rate in DS mice. The results suggest that early treatment with a partial TrkB receptor agonist may be a promising therapeutic approach to enhance PV interneuron function and reduce epileptogenesis and premature death in DS.

Astrocyte reactivity in a mouse model of SCN8A epileptic encephalopathy.

Objective SCN8A epileptic encephalopathy is caused predominantly by de novo gain‐of‐function mutations in the voltage‐gated sodium channel Nav1.6. The disorder is characterized by early onset of seizures and developmental delay. Most patients with SCN8A epileptic encephalopathy are refractory to current anti‐seizure medications. Previous studies determining the mechanisms of this disease have focused on neuronal dysfunction as Nav1.6 is expressed by neurons and plays a critical role in controlling neuronal excitability. However, glial dysfunction has been implicated in epilepsy and alterations in glial physiology could contribute to the pathology of SCN8A encephalopathy. In the current study, we examined alterations in astrocyte and microglia physiology in the development of seizures in a mouse model of SCN8A epileptic encephalopathy.MethodsUsing immunohistochemistry, we assessed microglia and astrocyte reactivity before and after the onset of spontaneous seizures. Expression of glutamine synthetase and Nav1.6, and Kir4.1 channel currents were assessed in astrocytes in wild‐type (WT) mice and mice carrying the N1768D SCN8A mutation (D/+).ResultsAstrocytes in spontaneously seizing D/+ mice become reactive and increase expression of glial fibrillary acidic protein (GFAP), a marker of astrocyte reactivity. These same astrocytes exhibited reduced barium‐sensitive Kir4.1 currents compared to age‐matched WT mice and decreased expression of glutamine synthetase. These alterations were only observed in spontaneously seizing mice and not before the onset of seizures. In contrast, microglial morphology remained unchanged before and after the onset of seizures.SignificanceAstrocytes, but not microglia, become reactive only after the onset of spontaneous seizures in a mouse model of SCN8A encephalopathy. Reactive astrocytes have reduced Kir4.1‐mediated currents, which would impair their ability to buffer potassium. Reduced expression of glutamine synthetase would modulate the availability of neurotransmitters to excitatory and inhibitory neurons. These deficits in potassium and glutamate handling by astrocytes could exacerbate seizures in SCN8A epileptic encephalopathy. Targeting astrocytes may provide a new therapeutic approach to seizure suppression.

Neurosteroids and Focal Epileptic Disorders.

Neurosteroids are a family of compounds that are synthesized in principal excitatory neurons and glial cells, and derive from the transformation of cholesterol into pregnenolone. The most studied neurosteroids—allopregnanolone and allotetrahydrodeoxycorticosterone (THDOC)—are known to modulate GABAA receptor-mediated transmission, thus playing a role in controlling neuronal network excitability. Given the role of GABAA signaling in epileptic disorders, neurosteroids have profound effects on seizure generation and play a role in the development of chronic epileptic conditions (i.e., epileptogenesis). We review here studies showing the effects induced by neurosteroids on epileptiform synchronization in in vitro brain slices, on epileptic activity in in vivo models, i.e., in animals that were made epileptic with chemoconvulsant treatment, and in epileptic patients. These studies reveal that neurosteroids can modulate ictogenesis and the occurrence of pathological network activity such as interictal spikes and high-frequency oscillations (80–500 Hz). Moreover, they can delay the onset of spontaneous seizures in animal models of mesial temporal lobe epilepsy. Overall, this evidence suggests that neurosteroids represent a new target for the treatment of focal epileptic disorders.

Spontaneous transitions to focal-onset epileptic seizures: A dynamical study.

Given the complex temporal evolution of epileptic seizures, understanding their dynamic nature might be beneficial for clinical diagnosis and treatment. Yet, the mechanisms behind, for instance, the onset of seizures are still unknown. According to an existing classification, two basic types of dynamic onset patterns plus a number of more complex onset waveforms can be distinguished. Here, we introduce a basic three-variable model with two time scales to study potential mechanisms of spontaneous seizure onset. We expand the model to demonstrate how coupling of oscillators leads to more complex seizure onset waveforms. Finally, we test the response to pulse perturbation as a potential biomarker of interictal changes.

Regulation of Synaptosomal GLT-1 and GLAST during Epileptogenesis

Astrocytes regulate extracellular glutamate homeostasis in the central nervous system through the Na+-dependent glutamate transporters glutamate transporter-1 (GLT-1) and glutamate aspartate transporter (GLAST). Impaired astrocyte glutamate uptake could contribute to the development of epilepsy but the regulation of glutamate transporters in epilepsy is not well understood. In this study, we investigate the expression of GLT-1 and GLAST in the mouse intrahippocampal kainic acid (IHKA) model of temporal lobe epilepsy (TLE). We used immunohistochemistry, synaptosomal fractionation and Western blot analysis at 1, 3, 7 and 30 days post-IHKA induced status epilepticus (SE) to examine changes in GLT-1 and GLAST immunoreactivity and synaptosomal expression during the development of epilepsy. We found a significant upregulation in GLT-1 immunoreactivity at 1 and 3 days post-IHKA in the ipsilateral dorsal hippocampus. However, GLT-1 immunoreactivity and synaptosomal protein levels were significantly downregulated at 7 days post-IHKA in the ipsilateral hippocampus, a time point corresponding to the onset of spontaneous seizures in this model. GLAST immunoreactivity was increased in specific layers at 1 and 3 days post-IHKA in the ipsilateral hippocampus. GLAST synaptosomal protein levels were significantly elevated at 30 days compared to 7 days post-IHKA in the ipsilateral hippocampus. Our findings suggest that astrocytic glutamate transporter dysregulation could contribute to the development of epilepsy.

The phenotypic landscape of a Tbc1d24 mutant mouse includes convulsive seizures resembling human early infantile epileptic encephalopathy.

Epilepsy, deafness, onychodystrophy, osteodystrophy and intellectual disability are associated with a spectrum of mutations of human TBC1D24. The mechanisms underlying TBC1D24-associated disorders and the functions of TBC1D24 are not well understood. Using CRISPR-Cas9 genome editing, we engineered a mouse with a premature translation stop codon equivalent to human S324Tfs*3, a recessive mutation of TBC1D24 associated with early infantile epileptic encephalopathy (EIEE). Homozygous S324Tfs*3 mice have normal auditory and vestibular functions but show an abrupt onset of spontaneous seizures at postnatal day 15 recapitulating human EIEE. The S324Tfs*3 variant is located in an alternatively spliced micro-exon encoding six perfectly conserved amino acids incorporated postnatally into TBC1D24 protein due to a micro-exon utilization switch. During embryonic and early postnatal development, S324Tfs*3 homozygotes produce predominantly the shorter wild-type TBC1D24 protein isoform that omits the micro-exon. S324Tfs*3 homozygotes show an abrupt onset of seizures at P15 that correlates with a developmental switch to utilization of the micro-exon. A mouse deficient for alternative splice factor SRRM3 impairs incorporation of the Tbc1d24 micro-exon. Wild-type Tbc1d24 mRNA is abundantly expressed in the hippocampus using RNAscope in situ hybridization. Immunogold electron microscopy using a TBC1D24-specific antibody revealed that TBC1D24 is associated with clathrin-coated vesicles and synapses of hippocampal neurons, suggesting a crucial role of TBC1D24 in vesicle trafficking important for neuronal signal transmission. This is the first characterization of a mouse model of human TBC1D24-associated EIEE that can now be used to screen for antiepileptogenic drugs ameliorating TBCID24 seizure disorders.

Chapter 3 - Presurgical intracranial investigations in epilepsy surgery

Identification and localization of the "epileptogenic process" in the brain of patients with drug-resistant epilepsy for surgical cure is the goal of presurgical investigations. Intracranial recordings are required when conflicting data between seizure clinical semiology and EEG prevent precise localization within one hemisphere or lateralization, when a visible lesion on MRI seems unrelated to the electroclinical data, or in MRI-negative cases. Two methods are currently used. The objective of the subdural grid electrocorticography with or without depth electrodes (SDG/DE) is the best possible identification of the area of onset of spontaneous seizures and localization of the eloquent cortex. The objective of stereoelectroencephalography (SEEG) is to define the epileptogenic zone (configured as a network) and its relation to an unmasked lesion. Two-dimensional (SDG) and three-dimensional (SEEG) brain sampling dictate different strategies for noninvasive presurgical phase I goals as well as for data analysis. SEEG must resolve several potential localization hypotheses in a manner that cannot be achieved with SDG. SDG operates through brain surface coverage, unlike SEEG, which samples networks. SDG estimates the extent of cortical resection through a lobar or sublobar localization of ictal onset and constraints from functional mapping. SEEG defines a tailored resection according to the results of anatomo-electro-clinical correlations in stereotaxic space that will guide the ablation of the epileptogenic zone. SEEG is currently expanding faster than SDG. The prerequisites (especially in the preimplantation hypothetical strategy) and technical tools (especially stimulation and functional mapping) in the two methods are very different. This chapter presents a comparative review of the rationale, indications, electrode implantation strategies, interpretation, and surgical decision making of these two approaches of presurgical evaluation for epilepsy surgery.

N-3 Docosapentaenoic acid-derived protectin D1 promotes resolution of neuroinflammation and arrests epileptogenesis.

Epilepsy therapy is based on drugs that treat the symptoms rather than the underlying mechanisms of the disease (epileptogenesis). There are no treatments for preventing seizures or improving disease prognosis, including neurological comorbidities. The search of pathogenic mechanisms of epileptogenesis highlighted that neuroinflammatory cytokines [i.e. interleukin-1β (IL-1β), tumour necrosis factor-α (Tnf-α)] are induced in human and experimental epilepsies, and contribute to seizure generation in animal models. A major role in controlling the inflammatory response is played by specialized pro-resolving lipid mediators acting on specific G-protein coupled receptors. Of note, the role that these pathways have in epileptogenic tissue remains largely unexplored. Using a murine model of epilepsy, we show that specialized pro-resolving mechanisms are activated by status epilepticus before the onset of spontaneous seizures, but with a marked delay as compared to the neuroinflammatory response. This was assessed by measuring the time course of mRNA levels of 5-lipoxygenase (Alox5) and 15-lipoxygenase (Alox15), the key biosynthetic enzymes of pro-resolving lipid mediators, versus Il1b and Tnfa transcripts and proteins. In the same hippocampal tissue, we found a similar delayed expression of two main pro-resolving receptors, the lipoxin A4 receptor/formyl peptide receptor 2 and the chemerin receptor. These receptors were also induced in the human hippocampus after status epilepticus and in patients with temporal lobe epilepsy. This evidence supports the hypothesis that the neuroinflammatory response is sustained by a failure to engage pro-resolving mechanisms during epileptogenesis. Lipidomic LC-MS/MS analysis showed that lipid mediator levels apt to resolve the neuroinflammatory response were also significantly altered in the hippocampus during epileptogenesis with a shift in the biosynthesis of several pro-resolving mediator families including the n-3 docosapentaenoic acid (DPA)-derived protectin D1. Of note, intracerebroventricular injection of this mediator during epileptogenesis in mice dose-dependently reduced the hippocampal expression of both Il1b and Tnfa mRNAs. This effect was associated with marked improvement in mouse weight recovery and rescue of cognitive deficit in the novel object recognition test. Notably, the frequency of spontaneous seizures was drastically reduced by 2-fold on average and the average seizure duration was shortened by 40% after treatment discontinuation. As a result, the total time spent in seizures was reduced by 3-fold in mice treated with n-3 DPA-derived protectin D1. Taken together, the present findings demonstrate that epilepsy is characterized by an inadequate engagement of resolution pathways. Boosting endogenous resolution responses significantly improved disease outcomes, providing novel treatment avenues.

Progressive changes in hippocampal cytoarchitecture in a neurodevelopmental rat model of epilepsy: implications for understanding presymptomatic epileptogenesis, predictive diagnosis, and targeted treatments

Epilepsies affect about 4% of the population and are frequently characterized by a prolonged “silent” period before the onset of spontaneous seizures. Most current animal models of epilepsy either involve acute seizure induction or kindling protocols that induce repetitive seizures. We have developed a rat model of epilepsy that is characterized by a slowly progressing series of behavioral abnormalities prior to the onset of behavioral seizures. In the current study, we further describe an accompanying progression of cytoarchitectural changes in the hippocampal formation. Groups of male and female SD rats received serial injections of a low dose of domoic acid (0.020 mg/kg) (or vehicle) throughout the second week of life. Postmortem hippocampal tissue was obtained on postnatal days 29, 64, and 90 and processed for glial fibrillary acidic protein (GFAP), NeuN, and calbindin expression. The data revealed no significant changes on postnatal day (PND) 29 but a significant increase in hilar NeuN-positive cells in some regions on PND 64 and 90 that were identified as ectopic granule cells. Further, an increase in GFAP positive cell counts and evidence of reactive astrogliosis was found on PND 90 but not at earlier time points. We conclude that changes in cellular expression, possibly due to on-going non-convulsive seizures, develop slowly in this model and may contribute to progressive brain dysfunction that culminates in a seizure-prone phenotype.

Biomarkers of Epileptogenesis: The Focus on Glia and Cognitive Dysfunctions.

The need to find measures that reliably predict the onset of epilepsy after injurious events or how the patient will respond to anti-seizure drugs led to intensive pre-clinical and clinical research to discover non-invasive biomarkers that could increase the sensitivity of existing clinical indicators. The use of experimental models of epileptogenesis and of drug-resistance is instrumental to select the most promising approaches to explore such biomarkers in the pre-clinical setting for further clinical validation. The approaches most frequently used to find clinically useful biomarkers of epileptogenesis include molecular brain imaging, EEG signal analysis and the measure of soluble molecules in biofluids which may reflect brain intrinsic events involved in epilepsy development. Among those, we focused our attention on proton magnetic resonance imaging (1H-MRS)-based analysis of astrocytic activation, and related blood biomarkers, since this cell population appears to be pivotally involved in various epileptogenesis processes triggered by differing insults. Moreover, we also investigated behavioral biomarkers by focusing on cognitive dysfunctions since this deficit represents a typical co-morbidity in epilepsy which may manifest even before the onset of spontaneous seizures. In this review article, we will report our recently published evidence supporting the utility of measuring astrocyte activation, the soluble molecules they release, and the associated cognitive deficits during epileptogenesis for early stratification of animals developing epilepsy. We will discuss the potential clinical translation of our findings for enriching the patient population in preventive clinical trials designed to study anti-epileptogenic treatments.

Enduring Memory Impairments Provoked by Developmental Febrile Seizures Are Mediated by Functional and Structural Effects of Neuronal Restrictive Silencing Factor.

In a subset of children experiencing prolonged febrile seizures (FSs), the most common type of childhood seizures, cognitive outcomes are compromised. However, the underlying mechanisms are unknown. Here we identified significant, enduring spatial memory problems in male rats following experimental prolonged FS (febrile status epilepticus; eFSE). Remarkably, these deficits were abolished by transient, post hoc interference with the chromatin binding of the transcriptional repressor neuron restrictive silencing factor (NRSF or REST). This transcriptional regulator is known to contribute to neuronal differentiation during development and to programmed gene expression in mature neurons. The mechanisms of the eFSE-provoked memory problems involved complex disruption of memory-related hippocampal oscillations recorded from CA1, likely resulting in part from impairments of dendritic filtering of cortical inputs as well as abnormal synaptic function. Accordingly, eFSE provoked region-specific dendritic loss in the hippocampus, and aberrant generation of excitatory synapses in dentate gyrus granule cells. Blocking NRSF transiently after eFSE prevented granule cell dysmaturation, restored a functional balance of γ-band network oscillations, and allowed treated eFSE rats to encode and retrieve spatial memories. Together, these studies provide novel insights into developing networks that underlie memory, the mechanisms by which early-life seizures influence them, and the means to abrogate the ensuing cognitive problems.SIGNIFICANCE STATEMENT Whereas seizures have been the central focus of epilepsy research, they are commonly accompanied by cognitive problems, including memory impairments that contribute to poor quality of life. These deficits often arise before the onset of spontaneous seizures, or independent from them, yet the mechanisms involved are unclear. Here, using a rodent model of common developmental seizures that provoke epilepsy in a subset of individuals, we identify serious consequent memory problems. We uncover molecular, cellular, and circuit-level mechanisms that underlie these deficits and successfully abolish them by targeted therapeutic interventions. These findings may be important for understanding and preventing cognitive problems in individuals suffering long febrile seizures.

Acute and spontaneous seizure onset zones in the intraperitoneal kainic acid model.

Hippocampal monitoring is often used in the intraperitoneal kainic acid (KA) seizure model for detection and quantification of early ictal activity. Here, we investigated extra-hippocampal seizure onset zones (SOZs) in this model. Eight male Sprague Dawley rats implanted with depth electrodes were continuously recorded during intraperitoneal KA injections until status epilepticus (SE) was induced. Another group of four rats was monitored chronically up to two weeks after emergence of spontaneous recurrent seizures. All rats had hippocampal electrodes. Other sampled brain regions included, among others, the claustrum, piriform cortex, and orbital cortex. Seizures recorded with video-EEG were visually analyzed. In the 58 seizures recorded during KA injections, the SOZ was extrahippocampal in 7 (12%), diffuse in 29 (50%), and hippocampal in 22 (38%). Of the 14 spontaneous seizures recorded, none were solely extrahippocampal, 10 (71%) were diffuse, and 4 (29%) were of hippocampal onset. All extra-hippocampal seizures propagated to the hippocampus within 4 to 50s (mean=14, n=7). No distinctive semiological manifestations correlated with the SOZs. We conclude that seizures can have multifocal SOZs in the KA model. This finding is important to consider when using this model, among other purposes, to screen for new therapies, study pharmacoresistance, or investigate comorbidities of epilepsy.

Neurosteroid-sensitive δ-GABAA receptors: A role in epileptogenesis?

We determined the role of the neurosteroid-sensitive δ subunit-containing γ-aminobutyric acid A receptors (δ-GABARs) in epileptogenesis. Status epilepticus (SE) was induced via lithium pilocarpine in adult rats, and seizures were assessed by continuous video-electroencephalography (EEG) monitoring. Finasteride was administered to inhibit neurosteroid synthesis. The total and surface protein expression of hippocampal δ, α4, and γ2 GABAR subunits was studied using biotinylation assays and Western blotting. Neurosteroid potentiation of the tonic currents of dentate granule cells (DGCs) was measured by whole-cell patch-clamp technique. Finally, the effects of inhibiting N-methyl-d-aspartate receptors (NMDARs) during SE on the long-term plasticity of δ-GABARs, neurosteroid-induced modulation of tonic current, and epileptogenesis were studied. The inhibition of neurosteroid synthesis 4 days after SE triggered acute seizures and accelerated the onset of chronic recurrent spontaneous seizures (epilepsy). The down-regulation of neurosteroid-sensitive δ-GABARs occurred prior to the onset of epilepsy, whereas an increased expression of the γ2-GABAR subunits occurred after seizure onset. MK801 blockade of NMDARs during SE preserved the expression of neurosteroid-sensitive δ-GABARs. NMDAR blockade during SE also prevented the onset of spontaneous seizures. Changes in neurosteroid-sensitive δ-GABAR expression correlated temporally with epileptogenesis. These findings raise the possibility that δ-GABAR plasticity may play a role in epileptogenesis.