Epileptogenesis is the complex process of the brain developing epileptic acitivity. Due to the role of glutamate and the hippocampus in synaptic plasticity a dysregulation in glutamate neurotransmission and hippocampal dysfunction are implicated in the process of epileptogenesis. However, the exact causal factors that promote epileptogenesis are unknown. We study presynaptic proteins that regulate glutamate neurotransmission and their role in epileptogenesis. The presynaptic protein, tomosyn, is believed to be a negative regulator of glutamate neurotransmission; however, no one has studied the effects of this protein on glutamate transmission in vivo. Furthermore, evidence suggests that mice lacking tomosyn have a kindling phenotype. Thus, in vivo glutamate recordings in mice lacking tomosyn have the potential to elucidate the exact role of tomosyn in glutamate neurotransmission and its potential relationship to epileptogenesis. Here we used biosensors to measure glutamate in the dentate gyrus (DG), CA3, and CA1 of the hippocampus in tomosyn wild-type (Tom+/+), heterozygous (Tom+/-), and knock out (Tom-/-) mice. We found that, in the DG, that glutamate release increases as tomosyn expression decreases across genotype. This suggests that tomosyn dysregulation in the DG leads to an increase in glutamate release, which may explain why these mice have an epileptogenic phenotype.

Mes travaux de thèse ont porté sur l’étude de deux modèles murins d’épilepsies génétiques de l’enfance liées à des mutations des canaux Nav1.1 (impliqués dans l’excitabilité des neurones inhibiteurs) : le Syndrome de Dravet (SD), une épilepsie pharmaco-résistante sévère, et l’Epilepsie Génétique avec Convulsions Fébriles Plus (EGCF+), présentant un phénotype plus modéré.Ils se sont décomposés en 3 axes : - La première partie mettant en évidence un phénomène d’épileptogenèse dans ces modèles murins.- La seconde permettant d’identifier des conditions expérimentales d’induction d'activités épileptiformes spécifiques du modèle murin du SD sur des tranches de cerveau.- La dernière consistant à mettre au point une stratégie pour réduire l’hyperexcitabilité cérébrale / During my thesis, I studied two murine models of childhood genetic epilepsies, caused by mutations of Nav1.1 channels (involved in the excitability of inhibitory neurons): Dravet Syndrome (DS), a severe and drug resistant epilepsy, and Genetic Epilepsy with Febrile Seizures Plus (GEFS+), characterized by a milder phenotype.My work is divided into three parts:- The first one revealed a process of epileptogenesis in these murine models.- In the second, I identified experimental conditions to induce epileptiform activities which are specific of the DS model in brain slices, which could allow pharmacological screens ex-vivo.- The third one was aimed at developing a new strategy to reduce cerebral hyperexcitability

The research of my thesis focused on the use of transcriptomic open discovery approaches to interrogate the molecular basis of two distinct yet related neurological disorders that are both associated with cognitive decline, Temporal Lobe Epilepsy and Alzheimer’s Disease. Interestingly, a potential role for compromised synaptogenesis early in disease was common to both, as was the direct role that neurons may play in brain inflammatory processes involving glia. Temporal lobe epilepsy (TLE) is a progressive disorder mediated by pathological changes in molecular cascades and hippocampal neural circuit remodeling that results in spontaneous seizures and cognitive dysfunction. Targeting these cascades may provide disease-modifying treatments for TLE patients. Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) inhibitors have emerged as potential disease-modifying therapies; however, a more detailed understanding of the contribution of JAK/STAT signaling to epileptogenesis is required to increase the potential therapeutic efficacy and reduce adverse effects associated with un-targeted JAK/STAT inhibition. With our collaborators, my lab developed a mouse line in which tamoxifen treatment conditionally abolishes STAT3 signaling from forebrain excitatory neurons (nSTAT3KO). Seizure frequency (continuous in vivo electroencephalography) and memory (contextual fear conditioning and motor learning) were analyzed in wildtype (Wt) and nSTAT3KO mice after intrahippocampal kainate (IHKA) injection as a model of TLE. Selective STAT3 KO in excitatory neurons reduced seizure progression and hippocampal memory deficits without reducing the extent of cell death or mossy fiber sprouting induced by IHKA injection. In my thesis, RNA was extracted from harvested hippocampi 24 h after IHKA and libraries were prepared for bulk RNA-sequencing (70–80 million reads/sample) using the NextSeq 500 Illumina system. 3190 genes were differentially expressed in Wt mice injected with KA vs saline (fold change |1.5|, FDR=<0.05). Ingenuity Pathway Analysis (IPA) revealed significant enrichment in 2 overarching sets of pathways: 1) those related to synaptic signaling and 2) those related to inflammation. As expected, many of the IHKA-induced genes were previously associated with epilepsy or seizure disorders (260 for Seizure Disorder, 267 for Epilepsy or Neurodevelopmental Disorder), and Seizure Disorder had the highest activation score in Neurological Disease based on gene expression patterns. Interestingly, a closer analysis of the IHKA-induced gene set revealed an enrichment of STAT3-associated genes (216), most of which were upregulated by IHKA. Compared to the 3190 Differentially Expressed Genes (DEGs) between IHKA and saline-injected Wt mice 24 hours after SE, more than half of these DEGs (1609) were rescued when comparing IHKA-injected nSTAT3KO mice and saline-injected Wt mice, indicating a significant rescue of gene expression when nSTAT3 is absent in excitatory neurons. While nSTAT3 KO influences the expression of genes in many different pathways, including the reversal of genes whose expression was inhibited in pathways of learning and memory by IHKA, the greatest surprise came from the predicted regulatory control over microglial function. nSTAT3KO mice displayed the greatest number of rescued DEGs compared to IHKA-injected WT mice in pathways that regulate inflammation and ion transport, and while inflammation was an expected response to IHKA, we were surprised to find evidence for its rescue in nSTAT3 KO mice. We also interrogated the expression of the Alzheimer’s disease genome as modeled using a rat model (TgF344-AD ) of familial AD that allows for behavioral and molecular characterization of AD, and expresses an endogenous pathogenic form of tau in addition to Abeta oligomers and plaques. AD is a neuropsychiatric disorder characterized initially by short term memory loss and disorientation, followed by declining cognitive functioning, and eventually, death. Widespread failure of 99% of AD drugs that make it to clinical trials has led to renewed interest in early signatures of disease in hopes of altering disease trajectory through early intervention. Key to such efforts is capturing a molecular window into AD at its earliest stages. The TgF344-AD rat shows overt pathology (including Aβ plaques, frank neuronal loss, and endogenous tau pathology) at 16 and 26 mo, but only to a very limited extent at 6 mo (Towne, 2013). Thus, in my thesis research, we set out to uncover any cell-type specific transcriptomic alterations that may be present in advance of major behavioral deficits or appearance of pathology, given that a strong body of literature suggests a long pre-symptomatic stage of illness in which subtle abnormalities may be present. 10x Genomics’ v3 gene expression assays were used to perform snRNA-seq on freshly dissected hippocampi from 6 mos, 9 mos and 19 mos littermate pairs of Tg and Wt rats (n=16 for 6 months and 9 months, with 8 for 19 months). ~2000 cells/subject were collected, and cDNA libraries were sequenced to a depth of ~120k reads/nuclei. Interestingly, data analysis revealed wide-scale gene changes in dentate granule cells (DGCs) and non-DGC excitatory neurons (Excit Ns) at 6 mos, suggestive of a significant decrease in synaptogenesis in Tg vs their Wt littermates, as well as small increases in cholesterol biosynthesis in the Tg rats in these cell types. By 9 months, some differentially expressed genes were observed across genotype in classes of glial cells, but the strongest impact on gene expression could still be seen in Excit Ns and DGCs, which continued to display evidence of decreased synaptogenesis, though to a lesser extent than at 6 mos. Interestingly, 9 mos Tg rats displayed an even stronger upregulation in genes related to cholesterol biosynthesis than 6 mos for both DGCs and Excit Ns. At 19 months, cholesterol and steroid biosynthesis were amongst the top biological pathways enriched for in Excit Ns and Inhibitory neurons of the Tg, to an even greater extent than changes in synaptogenesis. Altogether, our results suggest the transcriptional basis for a profound suppression of synapse formation or maintenance during early stages of illness in the TgF344-AD rat model, as well as abnormalities in neuronal cholesterol biosynthesis. Given that cholesterol is a key component of plasma membranes and lipid rafts, structures needed for the generation of new synapses and the stability of their receptor populations, it may be that deficiencies in the available cholesterol of Tg neuronal cells is leading to the impaired synaptogenesis in these cell types. Future work will focus on identifying whether these transcriptional alterations can be detected at even earlier time points, whether they are prescient for changes at the membrane in vivo that are correlated with memory impairment, and whether they are related to the alterations in the genome seen in our acquired epilepsy models, suggesting a common theme for the brain’s genomic response to injury of the hippocampus. / 2025-02-12T00:00:00Z

Orientador: Prof. Dr. Alexandre Hiroaki Kihara / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Neurociência e Cognição, 2016. / Status epilepticus (SE) is a clinical emergency that can lead to the development of temporal lobe epilepsy (TLE). The term epileptogenesis refers to the transformation of physiological neuronal networks into a dysfunctional state. In most patients presenting TLE, the development and maintenance of spontaneous seizures are linked with calcium (Ca+2)-dependent processes like neuronal loss, reactive gliosis and pathological neuronal plasticity. It has been shown that SE produces an increase in ryanodine receptor-dependent intracellular Ca+2 levels in hippocampal neurons, which remain elevated during the progression of the disease. In this context, the aim of this work was to investigate the effects of ryanodine receptors (RyRs) inhibition on the expression of important synaptic and plasticity-related proteins during the latent period of the pilocarpine model of TLE. First, we performed western blot and immunolabeling analyses in order to evaluate the pattern of distribution of the activity-regulated cytoskeleton-associated protein (ARC) in the rat hippocampus during the latent period. We observed decrease of the total protein levels 48 hours after SE, together with downregulation of its nuclear immunolabeling in granular cells of the dentate gyrus (DG). In addition, we observed the appearance of intense ARC immunoreactive neurons (IAINs) colocalizing mainly with excitatory neurons in CA1, CA3 and hilus. Intrahippocampal injections of the RyRs blocker dantrolene increased the total protein levels of the presynaptic protein synapsin I (SYN I) 48 hours after SE. We also observed up-regulation of SYN I and synaptophysin (SYP) in hippocampal regions known to receive important synaptic inputs. Finally, dantrolene showed neuroprotective effects by decreasing neuronal loss in CA1 and CA3 of experimental hippocampi. Our results suggest that ARC might be participating in the overall hippocampal reorganization and increase of excitability observed during epileptogenesis. In addition, RyRs may contribute to trigger the hippocampal neurodegeneration and synaptic alterations that lead to the development of acquired epilepsy.

Le cerveau est composé de réseaux de neurones interconnectés dont la mise en place au cours du développement cérébral est hautement régulée par des processus cellulaires, moléculaires et fonctionnels. Un dysfonctionnement de ces processus peut perturber l’établissement de ces réseaux et conduire à des pathologies neurologiques. L’épilepsie absence est une pathologie génétiquement déterminée qui apparait au cours de l’enfance. Les crises d’absences sont caractérisées par une altération de la conscience et par la présence de décharges de pointe-ondes (DPO) sur l’électroencéphalogramme initiées au sein d’un zone restreinte du cortex. Cependant, on sait peu de choses sur les mécanismes conduisant à la mise en place des décharges épileptiques récurrentes au cours de l’enfance (i.e. l’épileptogenèse). Nous avons fait l’hypothèse que des anomalies du processus de maturation cérébrale sont à l’origine de l’apparition des DPO.J’ai vérifié cette hypothèse chez un modèle génétique d’épilepsie absence, le rat GAERS. Dans un premier temps, j’ai étudié l’épileptogenèse du GAERS grâce à l’enregistrement in vivo du potentiel de champs local et de l’activité intracellulaire des neurones pyramidaux au niveau du site d’initiation des DPO, le cortex somatosensoriel (SoCX). Nous avons mis en évidence que les DPO se développent progressivement après la fin d’une période de maturation hautement sensible et malléable du SoCx (i.e. période critique). La maturation des décharges épileptiques consiste en une augmentation de leur fréquence, de leur durée et en l’évolution du motif de décharge jusqu’à l’âge adulte, période à laquelle ces paramètres atteignent une relative stabilité. De plus, ces changements sont associés à une altération graduelle des propriétés intrinsèques des neurones pyramidaux qui s’accompagne d’une augmentation progressive de la force de l’activité synaptique locale et d’une propension accrue des neurones du SoCx à générer des oscillations synchrones.Nous avons ensuite recherché les raisons de cette prédisposition anormale des neurones du SoCx à se synchroniser chez le GAERS. Dans ce but, nous avons cherché à mettre en évidence des anomalies de la maturation corticale au niveau de la structure et de l’organisation fonctionnelle du SoCx avant l’apparition des DPO. En combinant l’IRM, des marquages immunohistochimiques et le traçage rétrograde monosynaptique des connexions longue distance par le virus de la rage modifié, nous avons pu montrer qu’aucune anomalie globale du cerveau et du SoCx n’est présente chez le GAERS avant l’apparition des DPO. Afin de déterminer la présence d’anomalies fonctionnelles nous avons utilisé l’imagerie calcique biphoton et enregistré in vivo la dynamique de l’activité spontanée du réseau de neurones des couches 2-3 du SoCx. Chez le GAERS, nous avons mis en évidence que ces neurones sont plus actifs et dévoilent une organisation fonctionnelle différente de celle des animaux contrôles. Enfin, pour comprendre comment cette organisation fonctionnelle anormale est médiée, nous avons étudié la structure dendritique et synaptique du SoCx en combinant la microscopie électronique et la reconstruction morphologique de neurones. Nous avons mis en évidence un élargissement des épines dendritiques associé à un allongement de la densité post-synaptique au sein du SoCx chez le GAERS.L’ensemble de ces résultats démontrent la nature progressive du développement de l’épilepsie absence ainsi que l’existence d’anomalies de la maturation corticale qui affectent la structure et la fonction du réseau neuronal, avant l’apparition des crises épileptiques. Ces altérations constituent une prédisposition à l’établissement et l’évolution des DPO et sont une cible thérapeutique potentielle qui pourrait permettre de bloquer la mise en place des crises d’absences. / The brain is organized into several interconnected neuronal networks whose formation is highly regulated by cellular, molecular and functional processes. The dysfunction of these processes during brain development could disrupt neuronal circuit establishment and lead to neurological pathologies. Absence epilepsy is a genetically determined disease with a childhood onset. Absence seizures are characterized by an impairment of the consciousness associated on the electroencephalogram with spike and wave discharges (SWD). However, little is known about the mechanisms leading to the establishment of recurrent epileptic discharges (i.e. epileptogenesis). We hypothesized that SWD onset originates from an abnormal brain maturation.During my PhD, I examined this hypothesis in a recognized genetic model of absence epilepsy, the GAERS rat. First, I studied the epileptogenic process by recording in vivo the local field potential and the intracellular activities of pyramidal neurons in the initiating area of SWD, the somatosensory cortex (SoCx), at different post natal days in GAERS. We showed that SWD progressively developed after the end of a highly sensitive and plastic maturation period of the SoCx (i.e critical period). Afterward, epileptic discharges maturation consists in an increase of their duration, their number and in an evolution of the pattern reaching a relative stability at adulthood. Moreover, these changes are associated with a gradual abnormal alteration of the intrinsic properties of pyramidal neurons which is accompanied with a progressive increase in the strength of the local synaptic activity and a growing propensity of SoCx neurons to generate synchronized oscillations.Then, we explored the reasons for this abnormal susceptibility of SoCx neurons to be more synchronized in GAERS. We sought to bring to light anomalies of SoCx maturation at the structural and functional organization level prior to SWD onset in GAERS. Combining MRI, immunohistochemistry labeling and rabies-mediated retrograde monosynaptic tracing to reveal long-range connectivity, we showed that, prior to SWD onset, no brain and SoCx architecture abnormalities could be observed in GAERS. Then, using two photon calcium imaging we recorded in vivo the spontaneous activity of SoCx layers 2-3 neurons to evidence their functional organization. We found that these neurons were more active and unveiled a different functional organization in GAERS compared to control animals. Finally, to understand how is mediated this abnormal functional organization, we studied the dendritic and synaptic fine structure of SoCx neurons by combining electron microscopy and morphological neuron reconstruction. We highlighted an enlargement of the dendritic spines as well as an increase of the post-synaptic density length in the GAERS SoCx.Taken together, these findings showed the progressive nature of absence epilepsy development and the presence of abnormalities in the cortical maturation which affect the structure and the functional of the neuronal network the prior to SWD. These alterations constitute a breeding ground for the establishment and evolution of SWD. Future studies will aim at interfering with the epileptogenesis process should target these early alterations to stop seizure development.

<p>During cognitive tasks, synchronicity of neural activity varies and is correlated with performance. However, there may be an upper limit to normal synchronised activity – specifically, epileptogenic activity is characterized byexcess spiking at high synchronicity. An epileptic seizure has a complicated course of events and I therefore focused on the synchronised activity preceding a seizure (fast ripples). These high frequency oscillations (200–1000 Hz) have been identified as possible signature markers of epileptogenic activity and may be involved in generating seizures. Moreover, a range of ionic currents have been suggested to be involved in distinct aspects of epileptogenesis. Based on pharmacological and genetic studies, potassium currents have been implicated, in particular the transient A–type potassium channel (KA). Our first objective was to investigate if KA could suppress synchronized input while minimally affecting desynchronised input. The second objective was to investigate if KA could suppress fast ripple activity. To study this I use a detailed compartmental model of a hippocampal CA1 pyramidal cell. The ion channels were described by Hodgkin–Huxley dynamics.</p><p>The result showed that KA selectively could suppress highly synchronized input. I further used two models of fast ripple input and both models showed a strong reduction in the cellular spiking activity when KA was present. In an ongoing in vitro brain slice experiment our prediction from the simulations is being tested. Preliminary results show that the cellular response was reduced by 30 % for synchronised input, thus confirming our theoretical predictions. By suppressing fast ripples KA may prevent the highly synchronised spiking activity to spread and thereby preventing the seizure. Many antiepileptic drugs down regulate cell excitability by targeting sodium channels or GABA–receptors. These antiepileptic drugs affect the cell during normal brain activity thereby causing significant side effects. KA mainly suppresses the spiking activity when the cell is exposed to abnormally high synchronised input. An enhancement in the KA current might therefore be beneficial in reducing seizures while not affecting normal brain activity.</p>

Les études cliniques montrent que l’incidence des crises épileptiformes est la plus forte durant la période néonatale. Ces crises ont de nombreux facteurs étiologiques : un traumatisme crânien, des épisodes anoxo-ischémiques, des infections périnatales, des hémorragies intracrâniennes, des troubles métaboliques et de la fièvre... Ces crises per se peuvent entrainer des conséquences délétères à long terme. Notamment, l’hypothèse que la propagation des crises répétées vers des structures cérébrales naïves peut conduire à la formation d’un foyer épileptique secondaire qui génère des crises spontanées a été longtemps suggérée comme étant un mécanisme de base dans l’épilepsie humaine. Par conséquent, il est nécessaire de traiter efficacement les crises néonatales. Cependant, les traitements de premier choix comme le phénobarbital et le diazépam qui ont été développés pour traiter les crises chez l’adulte, sont souvent inefficaces chez les nouveau-nés et peuvent même aggraver les crises. Les mécanismes à l’origine de cette différence sont actuellement mal connus. Récemment, à l’aide d’une préparation développée dans le laboratoire composée des deux hippocampes néonataux interconnectés, il a été montré pour la première fois que des crises induites dans un hippocampe qui se propagent vers l’hippocampe controlatéral pouvaient conduire à la formation d’un foyer épileptique secondaire - foyer miroir (« seizure beget seizure »). Ce modèle a permis de montrer qu’un des mécanismes clés de la formation d’un foyer épileptique était l’augmentation permanente du chlore intracellulaire résultant en une action GABAergique excitatrice favorisant la genèse de crises spontanées. Déterminer les mécanismes à l’origine de l’épileptogenèse secondaire est d’une importance clinique majeure, et permettra de développer de nouvelles stratégies de prévention des effets pathologiques des crises.La première partie de ce travail a été de définir l’implication du co-transporteur de chlore NKCC1 dans la genèse de crises et l’épileptogenèse secondaire. Nous avons montré que le blocage de NKCC1, à l’aide d’outils pharmacologiques ou génétiques, ne prévient ni la formation d’un foyer miroir par des crises propagées ni l’augmentation permanente de chlore intracellulaire. Par conséquent, NKCC1 n’est ni nécessaire ni suffisant à induire ces modifications. Dans la deuxième partie, utilisant des outils électrophysiologiques et immunochimiques, nous apportons un faisceau d’évidences montrant que le co-transporteur de chlore KCC2 est internalisé et altéré fonctionnellement par des crises suggérant que l’accumulation de chlore résulte essentiellement de l’incapacité des neurones à évacuer le chlore. Dans la troisième partie nous avons étudié les effets du phénobarbital (PB) et du diazépam (DZP) sur la genèse de crises et l’épileptogenèse durant la période néonatale. En particulier, nous montrons que le PB, mais pas le DZP, bloque des crises initiales induites et prévient l’induction d’un foyer épileptique secondaire. Cette différence est due à un blocage partiel des récepteurs AMPA/KA par le PB. Cependant, une fois le foyer miroir établi, le PB comme le DZP aggravent les crises spontanées en exacerbant les effets excitateurs du GABA. Ces résultats montrent que l’histoire des crises détermine les effets du PB. En outre, le bumétanide, un antagoniste de NKCC1 qui réduit le chlore intracellulaire, améliore l’action du PB et bloque les crises spontanées. En conclusion, nos observations plaident fortement pour un traitement rapide des crises néonatales afin de protéger autant que faire les capacités du neurone à réguler le chlore intracellulaire. / Clinical studies show that children, especially neonates are in a much higher risk than adults to develop seizures. Such seizures in the brain may be provoked by different factors: tumor, infection, anoxia, fever, trauma, cysts, vascular malformations... Seizures in neonates are also often resistant to treatments and available antiepileptic drugs (AEDs) are inefficient or even provoke and aggravate neonatal seizures. A fundamental concept in epilepsy is that the seizures generated in epileptogenic regions propagate to the other brain structures even to the contralateral side and may develop permanent epileptic focus in the naïve brain structures – secondary epileptic focus. Consequently, it is necessary to treat the neonatal seizures. Diazepam (DZP) and phenobarbital (PB) are extensively used as first and second line drugs to treat acute seizures in neonates and their actions are thought to be mediated by increasing the actions of GABAergic signals. Yet, their efficacy is different and variable with occasional failure or even aggravation of recurrent seizures questioning whether other mechanisms are not involved in their actions. We studied these issues in the intact interconnected hippocampal preparation from neonatal rats and mice. Using this preparation and three-compartment chamber we induced seizures in one hippocampus that propagated to the contralateral one. The propagation of recurrent seizures transformed the contralateral hippocampus into independent epileptogenic focus – mirror focus (MF) - that was capable of generating spontaneous seizures (« seizure beget seizure »). The formation of MF is associated with a permanent increase of the intracellular concentration of chloride and a shift of the actions of GABA from inhibitory to excitatory. Therefore determining how secondary epileptogenesis is induced will have major clinical impact as it will enable to develop tools that prevent selectively the pathogenic seizures.At first, we have determined the impact and the contribution of chloride co-transporter NKCC1 in seizure generation and secondary epileptigenesis. We have shown that the pharmacologically or genetically blockade of NKCC1 did not prevent neither the generation nor propagation of evoked seizures nor formation of MF. However, in the isolated MF, bumetanide effectively blocked spontaneous epileptiform activity. Bumetanide partially reduced DFGABA and therefore the excitatory action of GABA in epileptic neurons. Therefore, bumetanide is a potent anticonvulsive agent although it cannot prevent formation of the epileptogenic MF.Second using different electrophysiological and immunochemistry approaches we have demonstrated that the accumulation of chloride and the excitatory actions of GABA in mirror foci neurons are mediated by NKCC1 chloride importer and by a downregulation and internalisation of the chloride exporter KCC2.Finally using our MF model we have compared the actions of PB and DZP on neonatal seizures. We have revealed that PB but not DZP dramatically reduced initial propagating seizures and prevented formation of epileptogenic MF. We show that PB in contrast to DZP has a highly specific action on AMPA/kainate receptor mediated currents. This action underlies an important difference between the two AEDs as in contrast to PB, DZP aggravates early seizures reflecting the advantage of PB over DZP to prevent secondary epileptogenesis. Yet, after repeated seizures, once an epileptogenic MF has been formed, this difference is abolished because of the strong excitatory actions of GABA. Therefore, the history of seizures prior to GABA acting AED treatment determines its effects and rapid treatment of severe potentially epileptogenic neonatal seizures is recommended to prevent secondary epileptogenesis associated with KCC2 down regulation.

In der Folge eines Status epilepticus entwickelt sich häufig eine chronische Epilepsie. In der vorliegenden Dissertation wurde die Fragestellung bearbeitet, ob ein Inhibitionsverlust im Gyrus dentatus Grundlage der Epileptogenese nach Status epilepticus ist. Ein selbst-erhaltender Status epilepticus (SSSE) wurde an erwachsenen Ratten durch elektrische Stimulation ausgelöst. Das Auftreten spontaner epileptischer Anfälle wurde im Verlauf von acht Wochen nach Status epilepticus zu drei Zeitpunkten (1, 4 und 8 Wochen) mittels Videoüberwachung erfasst. Zu denselben Zeitpunkten und vor Status epilepticus wurden elektrophysiologische Messungen im Gyrus dentatus durchgeführt. Die Aktivität der Prinzipalzellen des Gyrus dentatus unterliegt unter physiologischen Bedingungen einer ausgeprägten inhibitorischen Kontrolle. Durch Analyse von Doppelreizantworten wurden Veränderungen der Inhibition in dieser für die Epileptogenese relevanten Hirnstruktur beurteilt. Im Verlauf von acht Wochen nach SSSE entwickelte sich bei einem Großteil der Versuchstiere eine chronische Epilepsie. Zum spätesten Beobachtungszeitpunkt traten rekurrente spontane epileptische Anfälle bei 80 Prozent der Tiere auf. Die Inhibition im Gyrus dentatus war eine Woche nach Status epilepticus signifikant reduziert. Vier und acht Wochen nach SSSE zeigte sich eine zunehmende Wiederannäherung an die vor dem Status epilepticus erhobenen Messwerte, so dass von einem transienten Inhibitionsverlust im Gyrus dentatus nach Status epilepticus gesprochen werden kann. Zusammenfassend konnte in der Dissertation gezeigt werden, dass sich in der Folge eines Status epilepticus bei der Mehrzahl der Tiere eine chronische Epilepsie entwickelt. Der Inhibitionsverlust im Gyrus dentatus war zu einem Zeitpunkt am größten, da noch keine spontanen epileptischen Anfälle auftraten. Als sich bei den meisten Tieren eine chronische Epilepsie entwickelt hatte, war die Inhibition komplett wiederhergestellt. Daher ist ein Inhibitionsverlust im Gyrus dentatus nach einem Status epilepticus nicht der führende pathophysiologische Mechanismus für die Entwicklung einer chronischen Epilepsie. / Development of chronic epilepsy as a consequence of status epilepticus is a frequent clinical observation. The aim of this work was to test the hypothesis that epileptogenesis after status epilepticus depends on a loss of inhibitory function in the dentate gyrus. A self-sustaining status epilepticus (SSSE) was induced in rats by continuous electrical stimulation of the perforant path. The occurrence of spontaneous epileptic seizures was assessed by video monitoring 1, 4 and 8 weeks after SSSE. At the same time points and directly before SSSE, inhibition in the dentate gyrus was measured using a paired pulse paradigm. In this region, excitability of principal cells is under physiological conditions effectively controlled by the activity of inhibitory interneurons. In addition, the dentate gyrus is relevant for the process of epileptogenesis due to anatomical properties. In the time course after SSSE, the fraction of animals showing spontaneous epileptic seizures increased steadily reaching 80 % after eight weeks. One week after SSSE, inhibition in the dentate gyrus was significantly reduced. This loss proved to be transient, as inhibition recovered after 4 weeks and reached pre-status values after 8 weeks. In conclusion, the majority of animals developed chronic epilepsy as a consequence of status epilepticus. Loss of inhibition in the dentate gyrus was maximal while spontaneous seizures had not yet developed. Inhibition was normalized when most animals had become epileptic. Thus, loss of inhibition in the dentate gyrus following status epilepticus is not a decisive mechanism in the emergence of spontaneous seizures.

Η λειτουργική διαφοροποίηση κατά το διαφραγματοκροταφικό άξονα του ιπποκάμπου αφορά και την επιληψία. Χρησιμοποιώντας το μοντέλο ελεύθερο μαγνησίου και δυναμικά πεδίου παρατηρήσαμε πως οι επιληπτόμορφες εκφορτίσεις παρατηρούνταν πιο συχνά, είχαν μεγαλύτερη συχνότητα, διάρκεια και ένταση στις κοιλιακές τομές. Ο ανταγωνιστής των NMDA υποδοχέων AP5 μείωσε τη διάρκεια μόνο στις κοιλιακές τομές. Η προσθήκη του NMDA προκάλεσε εμμένουσες επιληπτόμορφες εκφορτίσεις στο 51% των κοιλιακών και το 9% των ραχιαίων τομών. Προτείνουμε πως οι υποδοχείς NMDA συμμετέχουν στη μεγαλύτερη ευπάθεια του κοιλιακού ιπποκάμπου τόσο στην έκφραση όσο και στη μακρόχρονη διατήρηση των επιληπτόμορφων εκφορτίσεων. Για να μελετήσουμε την επιληπτογένεση με άρση του αδενοσινεργικού τόνου, χρησιμοποιήσαμε πρωτόκολλα εκλεκτικού ή μη αποκλεισμού των αδενοσινεργικών υποδοχέων σε συνθήκες ελεύθερες μαγνησίου και καταγράψαμε αυθόρμητα ή προκλητά δυναμικά πεδίου στη CA3 σε κοιλιακές και ραχιαίες τομές. O αποκλεισμός του Α1 προκάλεσε επιληπτογένεση στο 31,13% των ραχιαίων και στο 52,76% των κοιλιακών τομών (P<0,05). Ο σύγχρονος αποκλεισμός του NMDA υποδοχέα αύξησε τα ποσοστά επιληπτογένεσης και στους δυο πόλους (76,38% στις ραχιαίες τομές vs 80,68% στις κοιλιακές τομές). Αυτή η NMDA-ανεξάρτητη επιληπτογένεση μειώθηκε σημαντικά με την προσθήκη του ανταγωνιστή των Α2 υποδοχέων κυρίως στις ραχιαίες τομές. O αποκλεισμός του Α1 υποδοχέα σε συνθήκες αποκλεισμού των NMDA υποδοχέων προκάλεσε παρόμοια αύξηση της κλίσης του fEPSP στις ραχιαίες τομές και στις κοιλιακές τομές. Ο επιπλέον αποκλεισμός των Α2 υποδοχέων επανέφερε την κλίση του fEPSP στο αρχικό της μέγεθος μόνο στις ραχιαίες τομές. Ο σύγχρονος αποκλεισμός των Α1 και Α2 υποδοχέων προκάλεσε επιληπτογένεση πρακτικά μόνο στις κοιλιακές τομές. H επιληπτογένεση αυτή ήταν μερικώς NMDA-εξαρτώμενη. Επιπλέον ενώ ο αποκλεισμός του Α1 προκάλεσε αύξηση της επιφάνειας της καμπύλης του fEPSP σε συνθήκες ελεύθερες μαγνησίου μόνο στις ραχιαίες τομές (96,15%), ο σύγχρονος αποκλεισμός των Α1 και Α2 υποδοχέων προκάλεσε αύξηση κατά 196,62% στις ραχιαίες τομές και 105,26% στις κοιλιακές τομές. Συμπεραίνουμε πως ο εκλεκτικός ή μη αποκλεισμός των υποδοχέων της αδενοσίνης προκαλεί διαφορετικά είδη επιληπτογένεσης που οφείλονται στις διαφορετικές δράσεις των υποδοχέων της αδενοσίνης και την ικανότητα του κοιλιακού ιπποκάμπου για ΝMDA-εξαρτώμενη επιληπτογένεση Χρησιμοποιώντας δυναμικά πεδίου σε κοιλιακές τομές και δυο μοντέλα επιληπτογένεσης παρατηρήσαμε πως οι σχετιζόμενες με τις επιληπτόμορφες εκφορτίσεις υψίσυχνες ταλαντώσεις και η διεγερτική νευροδιαβίβαση συμμεταβάλονται κατά τη διάρκεια της επιληπτογένεσης Παθολογικές υψίσυχνες ταλαντώσεις παρατηρήθηκαν πάντα στην NMDA-εξαρτημένη αλλά όχι και την NMDA-ανεξάρτητη επιληπτογένεση. Η διάρκεια των υψίσυχνων ταλαντώσεων συσχετίστηκε με τη διάρκεια των μεσοκριτικών εκφορτίσεων μόνο μετά την επαγωγή της επιληπτογένεσης Χρησιμοποιώντας ερεθισμό 100Hz και αυξημένη συγκέντρωση καλίου επάγαμε LTP με ερεθισμό των παράπλευρων κλάδων στη CA3 σε συνθήκες αποκλεισμού των υποδοχέων NMDA. Ο νέος τύπος του NMDA-ανεξάρτητου αυτού LTP παρουσίασε αργή ανάπτυξη στο χρόνο, δε μετέβαλε τη διευκόλυνση με σύζευξη παλμών και δεν επαγόταν με ταυτόχρονο αποκλεισμό των ευαίσθητων στη νιφεδιπίνη διαύλων ασβεστίου. Το μέγεθος του LTP ήταν σημαντικά μεγαλύτερο στις ραχιαίες τομές σε σχέση με τις κοιλιακές. / Functional segregation along the dorso-ventral axis of the hippocampus refers to epilepsy too. Using the model of magnesium-free medium and field recordings, single epileptiform discharges displayed higher incidence, rate, duration and intensity in ventral compared with dorsal rat hippocampal slices. The NMDA receptor antagonist AP5 shortened the discharges in ventral slices only. At 5 and 10μΜ of NMDA application 51% of the ventral but only 9% of the dorsal slices displayed persistent epileptiform discharges. We propose that the NMDA receptors contribute to the higher susceptibility of the ventral hippocampus to expression and long-term maintenance of epileptiform discharges. To study epileptogenesis following withdrawal of adenosinergic tone we used models of selective or non-selective blockade of adenosine receptors in magnesium-free medium and we recorded spontaneous or evoked field potentials in CA3 in dorsal as well as ventral slices. Blockade of A1 resulted in epileptogenesis in 31,13% of dorsal and in 52,76% of ventral slices used (P<0,05). NMDAR blockade increased epileptogenesis scores in both poles (76,38% in dorsal slices vs 80,68% in ventral slices). This NMDAR-dependent epileptogenesis was significantly aborted by blockade of A2R more in dorsal slices. Blockade of A1R under conditions of NMDAR blockade resulted to a similar increase of fEPSP slope in dorsal and ventral slices. The additional blockade of A2R decreased fEPSP slope to its original value in dorsal slices only. Simultaneous blockade of A1 and A2 receptors induced epileptogenesis practically in ventral slices only. This epileptogenesis was partially NMDA-dependent. Futrhermore A1R blockade resulted to an increase of fEPSP area under conditions of magnesium-free medium in dorsal slices only, whereas simultaneous blockade of both A1 and A2 receptors to an increase by 196,62% in dorsal slices and by 105,26% in ventral slices. We conclude that the selective or not blockade of adenosine receptors induces different kinds of epileptogenesis and this can be attributed to the different actions of adenosine receptors and the capability of ventral hippocampus to support NMDA-dependent epileptogenesis Employing field recordings from ventral hippocampal slices and two models of epileptogenesis, we found that HFOs associated with epileptiform bursts and excitatory synaptic transmission were co-modulated during epileptogenesis Pathological HFOs>200Hz were unequivocally present in persistent bursts induced by NMDA receptor-dependent but not NMDA receptor-independent mechanisms. The duration of pathological HFOs associated with persistent bursts but not of HFOs associated with bursts before the establishment of epileptogenesis was linearly and strongly correlated with the duration of bursts. Using 100Hz trains and medium with a higher concentration of potassium cations we induced LTP by stimulating associational/commissural fibers in CA3 region under conditions of NMDA receptor blockade. This new type of NMDAR-independent LTP displayed slow kinetics, did not change paired pulse facilitation and was prevented by simultaneous blockade of nifedipine-sensitive calcium channels. The incidence as well as the amplitude of LTP was greater in dorsal slices compared to ventral ones.

During cognitive tasks, synchronicity of neural activity varies and is correlated with performance. However, there may be an upper limit to normal synchronised activity – specifically, epileptogenic activity is characterized byexcess spiking at high synchronicity. An epileptic seizure has a complicated course of events and I therefore focused on the synchronised activity preceding a seizure (fast ripples). These high frequency oscillations (200–1000 Hz) have been identified as possible signature markers of epileptogenic activity and may be involved in generating seizures. Moreover, a range of ionic currents have been suggested to be involved in distinct aspects of epileptogenesis. Based on pharmacological and genetic studies, potassium currents have been implicated, in particular the transient A–type potassium channel (KA). Our first objective was to investigate if KA could suppress synchronized input while minimally affecting desynchronised input. The second objective was to investigate if KA could suppress fast ripple activity. To study this I use a detailed compartmental model of a hippocampal CA1 pyramidal cell. The ion channels were described by Hodgkin–Huxley dynamics. The result showed that KA selectively could suppress highly synchronized input. I further used two models of fast ripple input and both models showed a strong reduction in the cellular spiking activity when KA was present. In an ongoing in vitro brain slice experiment our prediction from the simulations is being tested. Preliminary results show that the cellular response was reduced by 30 % for synchronised input, thus confirming our theoretical predictions. By suppressing fast ripples KA may prevent the highly synchronised spiking activity to spread and thereby preventing the seizure. Many antiepileptic drugs down regulate cell excitability by targeting sodium channels or GABA–receptors. These antiepileptic drugs affect the cell during normal brain activity thereby causing significant side effects. KA mainly suppresses the spiking activity when the cell is exposed to abnormally high synchronised input. An enhancement in the KA current might therefore be beneficial in reducing seizures while not affecting normal brain activity.