The ATP-gated P2X7 receptor (P2X7R) is a non-selective cation channel which senses high extracellular ATP concentrations and has been suggested as a target for the treatment of neuroinflammation and neurodegenerative diseases. The use of P2X7R antagonists may therefore be a viable approach for treating CNS pathologies, including epileptic disorders. Recent studies showed anticonvulsant potential of P2X7R antagonists in certain animal models. To extend this work, we tested three CNS-permeable P2X7R blocker (Brilliant Blue G, AFC-5128, JNJ-47965567) and a natural compound derivative (tanshinone IIA sulfonate) in four well-characterized animal seizure models. In the maximal electroshock seizure threshold test and the pentylenetetrazol (PTZ) seizure threshold test in mice, none of the four compounds demonstrated anticonvulsant effects when given alone. Notably, in combination with carbamazepine, both AFC-5128 and JNJ-47965567 increased the threshold in the maximal electroshock seizure test. In the PTZ-kindling model in rats, useful for testing antiepileptogenic activities, Brilliant Blue G and tanshinone exhibited a moderate retarding effect, whereas the potent P2X7R blocker AFC-5128 and JNJ-47965567 showed a significant and long-lasting delay in kindling development. In fully kindled rats, the investigated compounds revealed modest effects to reduce the mean seizure stage. Furthermore, AFC-5128- and JNJ-47965567-treated animals displayed strongly reduced Iba 1 and GFAP immunoreactivity in the hippocampal CA3 region. In summary, our results show that P2X7R antagonists possess no remarkable anticonvulsant effects in the used acute screening tests, but can attenuate chemically-induced kindling. Further studies would be of interest to support the concept that P2X7R signalling plays a crucial role in the pathogenesis of epileptic disorders.

Entre 2 et 5% des enfants de 6 mois à 5 ans présentent au moins un épisode de Crise d’Épilepsie en contexte Fébrile (CEF). Bien que généralement bénignes, ces crises sont associées à un risque d’urgences neurologiques graves et curables dont l’élimination requiert la réalisation d’examens complémentaires douloureux et/ou irradiants. Actuellement, ce risque est évalué en fonction de trois facteurs : l’âge de l’enfant, le caractère simple ou complexe de la crise, et l’examen clinique.Cette thèse avait pour objectif de tester l’hypothèse que parmi les enfants consultant pour une CEF, seuls ceux avec un examen clinique anormal présentent un risque d’urgence neurologique grave et urgent. Pour ce faire, nous avons créé un outil informatique permettant une recherche exhaustive de cas parmi un million de dossiers médicaux informatisés dans sept services d’urgences pédiatriques entre 2007 et 2011. Nous avons alors identifié : les visites d’enfants présentant une CEF. Nous avons ensuite évalué le risque d’urgence neurologique grave et curable associé à ces visites, notamment lorsque l’examen clinique au décours était normal. Nous n’avons retrouvé aucune urgence neurologique grave et curable parmi les enfants consultant pour une CEF avec un examen clinique normal au décours, quels que soient l’âge et les caractéristiques de la crise. Ce travail de thèse associé aux données de la littérature confirme notre hypothèse et souligne la nécessité de recommandations quant à la prise en charge de ces enfants. Enfin, cette thèse constitue l’occasion de mener une réflexion méthodologique quant à l’utilisation de dossiers médicaux informatisés pour la recherche clinique. / Febrile seizures (FS) affect 2% to 5% of children aged 6 months to 5 years of age. Although FS are usually benign, they are associated with serious treatable neurological emergencies. Nowadays, three factors are used to evaluate this risk: the age of the child, whether the FS is simple or complex and the features of the clinical exam. The performance of a lumbar puncture and an emergent neuroimaging are required in order to rule out these emergencies. However, a lumbar puncture is painful and neuroimaging is irradiant. The objective of this thesis was to investigate the hypothesis that among children experiencing a FS, only those with an abnormal clinical exam are at risk of serious, treatable neurological emergencies. We first created an informatics tool in order to exhaustively search for cases among more than one million electronic medical records from seven pediatric emergency departments (PED) between 2007 and 2011. Then, we identified visits of children with a FS. Finally, we evaluated the proportion of serious, treatable neurological emergencies associated with these visits, and more specifically with visits of children with a normal clinical exam.We found no serious treatable neurological emergencies among children visiting the ED for a FS with a normal clinical exam, whatever the age and the features of the seizure were. The studies described in this thesis associated with the available data in the literature support our hypothesis and highlight the need of guidelines regarding the management of these children. Finally, this thesis gives us the opportunity to discuss some considerations on the use of electronic medical records for clinical research.

Neurons commonly have a primary cilium, which is a non-motile organelle extending from the centrosome into the extracellular space. In most brain regions, neuronal cilia are enriched in either somatostatin receptor type 3 (SstR3) or melanin concentrating hormone receptor type 1 (MCHR1), or both. The present immunohistochemical study provides novel evidence that primary cilia regulate neuronal excitability via G-protein coupled receptors (GPCRs), and that their identity is governed by brain region and by competition, both in adulthood and in postnatal development. The hippocampus, which is particularly vulnerable to seizures, has opposing gradients of SstR3(+) and MCHR1(+) ciliary GPCRs. We hypothesized that there is a competition between these two ciliary GPCRs, which might take place on any level from gene expression to presence in the cilium. We examined whether receptor colocalization occurs transiently in development before ciliary GPCR dominance is established in neurons in the CNS. In postnatal CA1 and CA3, the first GPCR to appear in cilia was the one that will dominate in adults: MCHR1 in CA1 and SstR3 in CA3. Some days later, the second GPCR was expressed along with the first; dual-receptor cilia were the exclusive type until single-receptor cilia emerged again around P14. Single-receptor cilia then increased in numbers through adulthood. By identifying ciliary receptors that modulate seizure activity in mice, the present study lays a foundation for therapeutic approaches to reduce neuronal excitotoxicity underlying cell death in epilepsy, CNS injury, and neurodegenerative diseases.

No description available.

<p dir="ltr">Recurrent stroke, infection, and seizure are some of the most common complications in stroke survivors. Recurrent stroke leads to death in 38.6% of survivors, and infections are the most common risk factor for seizures, with stroke survivors that experience an infection being at greater risk of experiencing a seizure. Two predictive models were generated, recurrent stroke and post-infection seizure, to determine stroke survivors at greatest risk to help providers focus on prevention in higher risk residents.</p><p dir="ltr">Predictive models were generated from a retrospective study of the Long-Term Care Minimum Data Set (MDS) 3.0 (2014-2018, n=262,301). Techniques included three data balancing methods (SMOTE for up sampling, ENN for down sampling, and SMOTEENN for up and down sampling) and three feature selection methods (LASSO, RFE, and PCA). The resulting datasets were then trained on four machine learning models (Logistic Regression, Random Forest, XGBoost, and Neural Network). Model performance was evaluated with AUC and accuracy, and interpretation used SHapley Addictive exPlanations.</p><p dir="ltr">Using data balancing methods improved the prediction performances of the machine learning models, but feature selection did not remove any features or affect performance. With all models having a high accuracy (78.6% to 99.9%), interpretation on all four models yielded the most holistic view. For recurrent stroke, SHAP values indicated that treatment combinations of occupational therapy, physical therapy, antidepressants, non-medical intervention for pain, therapeutic diet, anticoagulants, and diuretics contributed more to reducing recurrent stroke risk in the model when compared to individual treatments. For post-infection seizure, SHAP values indicated that therapy (speech, physical, occupational, and respiratory), independence (activities of daily living for walking, mobility, eating, dressing, and toilet use), and mood (severity score, anti-anxiety medications, antidepressants, and antipsychotics) features contributed the most. Meaning, stroke survivors who received fewer therapy hours, were less independent, and had a worse overall mood were at a greater risk of having a post-infection seizure.</p><p dir="ltr">The development of a tool to predict recurrent stroke and post-infection seizure in stroke survivors can be interpreted by providers to guide treatment and rehabilitation to prevent complications long-term. This promotes individualized plans that can increase the quality of resident care.</p>

This thesis deals with the problem of newborn seizre detection from the Electroencephalogram (EEG) signals. The ultimate goal is to design an automated seizure detection system to assist the medical personnel in timely seizure detection. Seizure detection is vital as neurological diseases or dysfunctions in newborn infants are often first manifested by seizure and prolonged seizures can result in impaired neuro-development or even fatality. The EEG has proved superior to clinical examination of newborns in early detection and prognostication of brain dysfunctions. However, long-term newborn EEG signals acquisition is considerably more difficult than that of adults and children. This is because, the number of the electrodes attached to the skin is limited by the size of the head, the newborns EEGs vary from day to day, and the newborns are reluctant of being in the recording situation. Also, the movement of the newborn can create artifact in the recording and as a result strongly affect the electrical seizure recognition. Most of the existing methods for neonates are either time or frequency based, and, therefore, do not consider the non-stationarity nature of the EEG signal. Thus, notwithstanding the plethora of existing methods, this thesis applies the discrete wavelet transform (DWT) to account for the non-stationarity of the EEG signals. First, two methods for seizure detection in neonates are proposed. The detection schemes are based on observing the changing behaviour of a number of statistical quantities of the wavelet coefficients (WC) of the EEG signal at different scales. In the first method, the variance and mean of the WC are considered as a feature set to dassify the EEG data into seizure and non-seizure. The test results give an average seizure detection rate (SDR) of 97.4%. In the second method, the number of zero-crossings, and the average distance between adjacent extrema of the WC of certain scales are extracted to form a feature set. The test obtains an average SDR of 95.2%. The proposed feature sets are both simple to implement, have high detection rate and low false alarm rate. Then, in order to reduce the complexity of the proposed schemes, two optimising methods are used to reduce the number of selected features. First, the mutual information feature selection (MIFS) algorithm is applied to select the optimum feature subset. The results show that an optimal subset of 9 features, provides SDR of 94%. Compared to that of the full feature set, it is clear that the optimal feature set can significantly reduce the system complexity. The drawback of the MIFS algorithm is that it ignores the interaction between features. To overcome this drawback, an alternative algorithm, the mutual information evaluation function (MIEF) is then used. The MIEF evaluates a set of candidate features extracted from the WC to select an informative feature subset. This function is based on the measurement of the information gain and takes into consideration the interaction between features. The performance of the proposed features is evaluated and compared to that of the features obtained using the MIFS algorithm. The MIEF algorithm selected the optimal 10 features resulting an average SDR of 96.3%. It is also shown, an average SDR of 93.5% can be obtained with only 4 features when the MIEF algorithm is used. In comparison with results of the first two methods, it is shown that the optimal feature subsets improve the system performance and significantly reduce the system complexity for implementation purpose.

The goal of this research was to determine the procedures used for conducting a search and seizure in a tax-related offence in terms of the Criminal Procedure Act, Act 51 of 1977. Aspects that cause problems for the South African Revenue Service (SARS) investigators are the application for a search warrant and the activities that take place before, during and after the search and seizure. An introduction, definition and explanation are given of certain key concepts such as forensic and criminal investigations, as well as their objectives and purpose. The various search methods are also discussed and explained as are the chain of custody and evidence in general. A large part of this research deals with the legal requirements for a search and seizure in a tax-related offence and encompasses issues such as the procedures for obtaining a search warrant, pre-raid briefing, conducting the search, and the seizing of, marking, storage and disposal of documents. The findings of the research are discussed and recommendations subsequently made regarding the shortcomings identified. The findings that were made related to the process and procedure to obtain a search warrant, the actual execution of a search and seizure and the legislation that authorises searches and seizures in taxrelated offences. Further findings were made in respect of the mandate of SARS criminal investigators to investigate, the admissibility of evidence obtained from a search and seizure and the marking, recording, storage and disposal of seized items. Recommendations were made regarding training, improved communication and skills transfer to address the shortcomings identified. / Police Practice / (M.Tech. (Forensic investigation))

Thesis (MComm)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Section 74D of the Income Tax Act No 58 of 1962 (the Act) grants the power of search and seizure to the South African Revenue Service, the basic underlying principle being that the Commissioner has to obtain a warrant from a judge prior to a search and seizure operation. The previous section 74(3) of the Act provided that the Commissioner was allowed himself to authorise and conduct a search and seizure operation without the requirement of a warrant. Section 74D of the Act was recently reviewed and the Tax Administration Bill (the TAB) contains the new provisions on search and seizure that will replace section 74D of the Act. In this assignment, the concept of search and seizure was examined by considering the cases, academic writing and other material on the topic. The objectives were to analyse the development of search and seizure in South African income tax law, to provide a basic understanding of the warranted and warrantless search and seizure provisions of the Act and the TAB, to determine their constitutionality and to determine the remedies available to a taxpayer who has been subject to a search and seizure. It was found that search and seizure has developed from warrantless under the previous section 74(3) of the Act into the requirement of a warrant under section 74D of the Act into a combination of both under the TAB. The concept of an ex parte application was analysed, which was shown to be permissible in certain circumstances under section 74D of the Act, while it is now compulsory in terms of the TAB. It was shown that the TAB closed the lacuna in the Act relating to the validity period of a warrant before it has been executed. It was, however, concluded, regarding whether a warrant expires when exercised or whether the same warrant can be used again to conduct a second search and seizure, that the position is not quite certain in terms of the Act and the TAB. It was found that there is no defined meaning of the reasonable grounds criterion, which is often required to be met in terms of the Act and the TAB, but that anyone that has to comply with the criterion must be satisfied that the grounds in fact exist objectively. The new warrantless search and seizure provisions of the TAB were analysed. It was established that warrantless search and seizure provisions are not uncommon in other statutes, but that the content thereof often differs. The new warrantless provisions were compared to the warrantless search and seizure provisions of, inter alia, the Competition Act No 89 of 1998 (the Competition Act), and it was found that the warrantless TAB provisions are not in all respects as circumscribed as those of the Competition Act and recommendations for counterbalances were made. It was concluded that the warranted search and seizure provisions of the Act and the TAB should be constitutionally valid but that the constitutionality of the new warrantless provisions of the TAB is not beyond doubt. It was furthermore found that the remedies at the disposal of a taxpayer who has been subject to a search and seizure should indeed be sufficient, but that there are no remedies available to a taxpayer to prevent injustice or harm. / AFRIKAANSE OPSOMMING: Artikel 74D van die Inkomstebelastingwet No 58 van 1962, (die Wet) verleen aan die Suid-Afrikaanse Inkomstediens die mag van deursoeking en beslaglegging, die grondliggende beginsel synde dat die Kommissaris ’n lasbrief van ’n regter moet verkry voor die deursoeking en beslaglegging kan plaasvind. Die vorige artikel 74(3) van die Wet het bepaal dat die Kommissaris self ’n deursoeking en beslaglegging kon magtig en uitvoer sonder die vereiste van ’n lasbrief. Artikel 74D van die Wet is onlangs hersien en die nuwe Belastingadministrasie-wetsontwerp (BAW) bevat die nuwe bepalings oor deursoeking en beslaglegging wat artikel 74D van die Wet sal vervang. In hierdie werkstuk is die konsep van deursoeking en beslaglegging ondersoek deur oorweging van die hofsake, akademiese skrywe en ander materiaal oor die onderwerp. Die doelstellings was om die ontwikkeling van deursoeking en beslaglegging in die Suid-Afrikaanse inkomstebelastingreg te ontleed, om ’n basiese begrip van die bepalings in die Wet en die BAW oor deursoeking en beslaglegging met en sonder ’n lasbrief te verskaf, om die grondwetlikheid daarvan te bepaal en om die remedies te bepaal wat beskikbaar is vir ’n belastingpligtige wat onderworpe was aan deursoeking en beslaglegging. Daar is bevind dat deursoeking en beslaglegging ontwikkel het vanaf sonder ’n lasbrief ingevolge die vorige artikel 74(3) van die Wet tot die vereiste van ’n lasbrief ingevolge artikel 74D van die Wet tot die kombinasie van albei ingevolge die BAW. Die konsep van ’n ex parte-aansoek is ontleed, en dit blyk in sekere omstandighede ingevolge artikel 74D van die Wet toelaatbaar te wees, terwyl dit nou ingevolge die BAW verpligtend is. Daar is aangedui dat die BAW die lacuna in die Wet oor die geldigheidsperiode van ’n lasbrief voordat dit uitgevoer is, verwyder het. Daar is egter bevind, rakende die vraag of ’n lasbrief verval wanneer dit uitgevoer word en of dieselfde lasbrief weer gebruik kan word om ’n tweede deursoeking en beslaglegging uit te voer, dat daar nie sekerheid ingevolge die Wet of die BAW bestaan nie. Daar is bevind dat daar geen gedefinieerde betekenis vir die kriterium van redelike gronde is nie, waaraan dikwels ingevolge die Wet en die BAW voldoen moet word, maar dat enigiemand wat aan die kriterium moet voldoen tevrede moet wees dat die gronde inderwaarheid objektief bestaan. Die nuwe bepalings van die BAW oor deursoeking en beslaglegging sonder ’n lasbrief is ondersoek. Daar is vasgestel dat bepalings oor deursoeking en beslaglegging sonder ’n lasbrief nie ongewoon is in ander wette nie, maar dat die inhoud daarvan dikwels verskil. Die nuwe bepalings oor deursoeking en beslaglegging sonder ’n lasbrief is vergelyk met die bepalings oor deursoeking en beslaglegging sonder ’n lasbrief van, inter alia, die Mededingingswet No 89 van 1998 (die Mededingingswet), en daar is bevind dat die BAW-bepalings oor deursoeking en beslaglegging sonder ’n lasbrief nie in alle opsigte so afgebaken is soos dié van die Mededingingswet nie en voorstelle vir teenwigte is gemaak. Die gevolgtrekking is gemaak dat die bepalings oor deursoeking en beslaglegging met ’n lasbrief van die Wet en die BAW grondwetlik geldig behoort te wees, maar dat die grondwetlikheid van die nuwe bepalings van die BAW oor deursoeking en beslaglegging sonder ’n lasbrief nie onweerlegbaar is nie. Daar is verder bevind dat die remedies tot die beskikking van ’n belastingpligtige wat onderworpe was aan deursoeking en beslaglegging inderdaad genoegsaam behoort te wees, maar dat daar geen remedies aan ’n belastingpligtige beskikbaar is om ongeregtigheid of skade te voorkom nie.

La sclérose tubéreuse de Bourneville (STB) est une maladie génétique et multi-systémique à transmission autosomique dominante due à des mutations d’un gène TSC1 ou TSC2 qui codent respectivement pour hamartine et tuberine ayant une action inhibitrice sur la voie de signalisation mTOR. L’épilepsie précoce et pharmacorésistante est la manifestation neurologique la plus fréquente et la plus délétère de la STB. Elle débute souvent dans la première année de vie par des spasmes infantiles qui évoluent avec l’âge et en absence de traitement vers des crises toniques ou tonico-cloniques. Bien que les crises soient supposées être générées dans des tubers corticaux, les mécanismes de l’épilepsie ne sont pas bien élucidés et le traitement reste souvent inefficace. Des études morphologiques ont montré une altération de l’expression ARNm des récepteurs au glutamate dans les cellules géantes et les neurones dysplasiques des tubers, mais leur implication fonctionnelle restait à montrer. Les différentes sous-unités NMDA ont une expression âge-dépendante et région-spécifique, les plus grands changements survenant au début de la vie quand l’épilepsie de la STB apparaît. Ce travail avait pour but d’étudier à l’aide de méthodes électrophysiologiques in vitro et in vivo l’expression fonctionnelle des sous-unités NMDA aberrantes et de déterminer leur rôle dans l’épileptogènese chez les souris hétérozygotes Tsc1+/- et sur le tissu humain STB post-opératoire. Nous avons pu démontrer que : i) Les souris hétérozygotes pour le gène Tsc1 sont spontanément épileptiques in vivo et in vitro dans une courte fenêtre dévelopmentale de P9 à P18. ii) Elles présentent une altération d’expression des récepteurs NMDA couche-spécifique et mTOR dépendante avec une surexpression des sous-unités GluN2C/D dans la couche 4 et 2/3 et GluN2B dans les couches 2/3. Cette expression anormale est prévenue par l’administration d’un inhibiteur de la voie mTOR, la rapamycine. iii) Les mêmes altérations d’expression des récepteurs NMDA, sont montrées sur les tissus post-opératoires, non seulement de tubers de STB mais aussi des dysplasies corticales focales (DCF), ces deux malformations ayant des similarités étiologiques et physiopathologiques. iv) La RT-PCR quantitative confirme une expression excessive de GluN2C dans le cortex de souris Tsc1+/- et sur le tissu humain des tubers et DCF. v) Les décharges épileptiques chez la souris Tsc1+/- sont générées dans la couche granulaire 4 du cortex avant de se propager vers les couches superficielles et les couches profondes, empruntant ainsi les microcircuits corticaux. vi) L’expression excessive de la sous-unité GluN2C dans le cortex contribue à l’hyperexcitabilité neuronale chez la souris Tsc1+/- et sur des tissus humains de tubers et de DCF puisque les crises et les décharges sont bloquées par les antagonistes sélectifs de GluN2C/D. vii) Les crises chez la souris Tsc1+/- suivent une séquence âge-dépendante évoluant du type «spasms-like» vers «tonic-clonic like», rappelant celle de l’épilepsie humaine, avec deux pics de haute incidence de crises à P13 et P16 correspondant chez l’homme respectivement l’âge des spasmes infantiles et celui des crises toniques. L’évolution avec l’âge du délai de propagation inter-hémisphérique pourrait contribuer à ce changement de types de crises. Ces résultats montrent donc pour la première fois qu’une happloinsuffisance pour le gène Tsc1 chez les souris Tsc1+/- sans tubers suffit à produire une altération de l’expression des récepteurs NMDA de manière mTOR dépendante et contribuer ainsi à l’épileptogènese dans la STB. La souris Tsc1+/- est le premier modèle génétique sans anomalies morphologiques présentant une épilepsie spontanée qui évolue des spasmes vers des crises toniques et tonico-cloniques. Néanmoins cette épilepsie diffère de l’épilepsie humaine de la STB par l’absence de crises focales et de pharmacorésistance, ce qui pourrait être expliqué par l’absence de tubers chez la souris Tsc1+/-. (...) / Tuberous sclerosis complex (TSC) is a genetic multisystemic disease with autosomal dominant transmission due to mutations in a gene TSC1 or TSC2 respectively which encode hamartin and tuberin proteins having an inhibitory action on the mTOR signaling pathway. Early refractory epilepsy is the most common and most deleterious neurological manifestation. The epilepsy often begins in the first year of life by infantile spasms that change in the lack of treatment to tonic or tonic-clonic seizures in age-dependent manner. Although seizures are thought to be generated in cortical tubers, epilepsy mechanisms are not well understood and treatment is often ineffective. Morphological studies showed the altered expression of glutamate receptor mRNA in the giant cells and dysplastic neurons of tubers, but their functional involvement remains unknown. The different NMDA subunits have an age-dependent and region-specific expression, the greatest changes occurring early in life when the TSC epilepsy appears. This work aimed to study the functional expression of aberrant NMDA subunits expression and their role in the epileptogenesis in heterozygous Tsc1+/- mice and post-surgical human tissue of TSC patients using in vitro and in vivo electrophysiological methods. The study reveal that: i) Heterozygous tuber-free Tsc1+/- mice show spontaneous epilepsy in vivo and in vitro in a short developmental window from P9 to P18. ii) These mice exhibit an altered NMDA receptor expression in mTOR dependent and layer-specific manner with GluN2C/D subunits overexpression in layers 4 and 2/3, and GluN2B ovexpression in layers 2/3. This abnormal NMDA receptors expression is prevented by the administration of an mTOR inhibitor, rapamycin. iii) The same alterations of NMDA receptors’ expression are shown in post-surgical tissues not only in tubers from TSC patients, but also in focal cortical dysplasia (FCD), these two malformations sharing etiological and pathophysiological similarities. iv) Quantitative RT-PCR confirms the excessive GluN2C subunit expression in Tsc1+/- mouse cortex and human tissue of tubers and DCF. v) Epileptic discharges in Tsc1+/- mice are generated in the granular layer 4 of the cortex before spreading to the superficial and then to deep layers, thus borrowing the cortical microcircuits. vi) Excessive expression of GluN2C subunit in the cortex contributes to neuronal hyperexcitability in Tsc1+/- mice, as well as in human tubers and DCF tissues, since epileptic discharges are blocked by selective GluN2C/D antagonists. vii) Seizures in Tsc1+/- mice follow the age-dependent sequence, evolving from "spasms-like" to "tonic-clonic like" thus reminding the human epilepsy, with two peaks of highest seizure incidence at P13 and P16 corresponding respectively to age of infantile spasms and of tonic seizures in human. The age-dependent evolution of interhemispheric propagation delay could contribute to this change in seizure type. These results show for the first time that TSC1 happloinsuffisancy in tuber-free Tsc1+/- mice is sufficient to produce an alteration in NMDA receptor expression in an mTOR dependent manner, and thus contributes to epileptogenesis in TSC. The Tsc1+/- mouse line is the first genetic model of TSC without morphological abnormalities presenting with early spontaneous seizures which evolves from “spasms-like” to “tonic-clonic like” seizures. However, the epilepsy in Tsc1+/- mice differs from human TSC epilepsy by the absence of focal seizures and of drug-resistance. Both could be explained by the lack of tubers in the Tsc1+/- mice. It remains to determine whether the expression of GluN2C subunit is also transitional in Tsc1+/- mice and whether other factors contribute to determine the age-dependent epilepsy. This study opens new therapeutic perspectives of TSC epilepsy targeting GluN2C subunit of NMDA receptors.

La sclérose tubéreuse de Bourneville (STB) est une maladie génétique et multi-systémique à transmission autosomique dominante due à des mutations d’un gène TSC1 ou TSC2 qui codent respectivement pour hamartine et tuberine ayant une action inhibitrice sur la voie de signalisation mTOR. L’épilepsie précoce et pharmacorésistante est la manifestation neurologique la plus fréquente et la plus délétère de la STB. Elle débute souvent dans la première année de vie par des spasmes infantiles qui évoluent avec l’âge et en absence de traitement vers des crises toniques ou tonico-cloniques. Bien que les crises soient supposées être générées dans des tubers corticaux, les mécanismes de l’épilepsie ne sont pas bien élucidés et le traitement reste souvent inefficace. Des études morphologiques ont montré une altération de l’expression ARNm des récepteurs au glutamate dans les cellules géantes et les neurones dysplasiques des tubers, mais leur implication fonctionnelle restait à montrer. Les différentes sous-unités NMDA ont une expression âge-dépendante et région-spécifique, les plus grands changements survenant au début de la vie quand l’épilepsie de la STB apparaît. Ce travail avait pour but d’étudier à l’aide de méthodes électrophysiologiques in vitro et in vivo l’expression fonctionnelle des sous-unités NMDA aberrantes et de déterminer leur rôle dans l’épileptogènese chez les souris hétérozygotes Tsc1+/- et sur le tissu humain STB post-opératoire. Nous avons pu démontrer que : i) Les souris hétérozygotes pour le gène Tsc1 sont spontanément épileptiques in vivo et in vitro dans une courte fenêtre dévelopmentale de P9 à P18. ii) Elles présentent une altération d’expression des récepteurs NMDA couche-spécifique et mTOR dépendante avec une surexpression des sous-unités GluN2C/D dans la couche 4 et 2/3 et GluN2B dans les couches 2/3. Cette expression anormale est prévenue par l’administration d’un inhibiteur de la voie mTOR, la rapamycine. iii) Les mêmes altérations d’expression des récepteurs NMDA, sont montrées sur les tissus post-opératoires, non seulement de tubers de STB mais aussi des dysplasies corticales focales (DCF), ces deux malformations ayant des similarités étiologiques et physiopathologiques. iv) La RT-PCR quantitative confirme une expression excessive de GluN2C dans le cortex de souris Tsc1+/- et sur le tissu humain des tubers et DCF. v) Les décharges épileptiques chez la souris Tsc1+/- sont générées dans la couche granulaire 4 du cortex avant de se propager vers les couches superficielles et les couches profondes, empruntant ainsi les microcircuits corticaux. vi) L’expression excessive de la sous-unité GluN2C dans le cortex contribue à l’hyperexcitabilité neuronale chez la souris Tsc1+/- et sur des tissus humains de tubers et de DCF puisque les crises et les décharges sont bloquées par les antagonistes sélectifs de GluN2C/D. vii) Les crises chez la souris Tsc1+/- suivent une séquence âge-dépendante évoluant du type «spasms-like» vers «tonic-clonic like», rappelant celle de l’épilepsie humaine, avec deux pics de haute incidence de crises à P13 et P16 correspondant chez l’homme respectivement l’âge des spasmes infantiles et celui des crises toniques. L’évolution avec l’âge du délai de propagation inter-hémisphérique pourrait contribuer à ce changement de types de crises. Ces résultats montrent donc pour la première fois qu’une happloinsuffisance pour le gène Tsc1 chez les souris Tsc1+/- sans tubers suffit à produire une altération de l’expression des récepteurs NMDA de manière mTOR dépendante et contribuer ainsi à l’épileptogènese dans la STB. La souris Tsc1+/- est le premier modèle génétique sans anomalies morphologiques présentant une épilepsie spontanée qui évolue des spasmes vers des crises toniques et tonico-cloniques. Néanmoins cette épilepsie diffère de l’épilepsie humaine de la STB par l’absence de crises focales et de pharmacorésistance, ce qui pourrait être expliqué par l’absence de tubers chez la souris Tsc1+/-. (...) / Tuberous sclerosis complex (TSC) is a genetic multisystemic disease with autosomal dominant transmission due to mutations in a gene TSC1 or TSC2 respectively which encode hamartin and tuberin proteins having an inhibitory action on the mTOR signaling pathway. Early refractory epilepsy is the most common and most deleterious neurological manifestation. The epilepsy often begins in the first year of life by infantile spasms that change in the lack of treatment to tonic or tonic-clonic seizures in age-dependent manner. Although seizures are thought to be generated in cortical tubers, epilepsy mechanisms are not well understood and treatment is often ineffective. Morphological studies showed the altered expression of glutamate receptor mRNA in the giant cells and dysplastic neurons of tubers, but their functional involvement remains unknown. The different NMDA subunits have an age-dependent and region-specific expression, the greatest changes occurring early in life when the TSC epilepsy appears. This work aimed to study the functional expression of aberrant NMDA subunits expression and their role in the epileptogenesis in heterozygous Tsc1+/- mice and post-surgical human tissue of TSC patients using in vitro and in vivo electrophysiological methods. The study reveal that: i) Heterozygous tuber-free Tsc1+/- mice show spontaneous epilepsy in vivo and in vitro in a short developmental window from P9 to P18. ii) These mice exhibit an altered NMDA receptor expression in mTOR dependent and layer-specific manner with GluN2C/D subunits overexpression in layers 4 and 2/3, and GluN2B ovexpression in layers 2/3. This abnormal NMDA receptors expression is prevented by the administration of an mTOR inhibitor, rapamycin. iii) The same alterations of NMDA receptors’ expression are shown in post-surgical tissues not only in tubers from TSC patients, but also in focal cortical dysplasia (FCD), these two malformations sharing etiological and pathophysiological similarities. iv) Quantitative RT-PCR confirms the excessive GluN2C subunit expression in Tsc1+/- mouse cortex and human tissue of tubers and DCF. v) Epileptic discharges in Tsc1+/- mice are generated in the granular layer 4 of the cortex before spreading to the superficial and then to deep layers, thus borrowing the cortical microcircuits. vi) Excessive expression of GluN2C subunit in the cortex contributes to neuronal hyperexcitability in Tsc1+/- mice, as well as in human tubers and DCF tissues, since epileptic discharges are blocked by selective GluN2C/D antagonists. vii) Seizures in Tsc1+/- mice follow the age-dependent sequence, evolving from "spasms-like" to "tonic-clonic like" thus reminding the human epilepsy, with two peaks of highest seizure incidence at P13 and P16 corresponding respectively to age of infantile spasms and of tonic seizures in human. The age-dependent evolution of interhemispheric propagation delay could contribute to this change in seizure type. These results show for the first time that TSC1 happloinsuffisancy in tuber-free Tsc1+/- mice is sufficient to produce an alteration in NMDA receptor expression in an mTOR dependent manner, and thus contributes to epileptogenesis in TSC. The Tsc1+/- mouse line is the first genetic model of TSC without morphological abnormalities presenting with early spontaneous seizures which evolves from “spasms-like” to “tonic-clonic like” seizures. However, the epilepsy in Tsc1+/- mice differs from human TSC epilepsy by the absence of focal seizures and of drug-resistance. Both could be explained by the lack of tubers in the Tsc1+/- mice. It remains to determine whether the expression of GluN2C subunit is also transitional in Tsc1+/- mice and whether other factors contribute to determine the age-dependent epilepsy. This study opens new therapeutic perspectives of TSC epilepsy targeting GluN2C subunit of NMDA receptors.