Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Epilepsy is one of the most common neurologic disorders. It has been suggested that seizures may be facilitaded by inflammation. PGE2 is one of the most important inflammatory mediators, and facilitates pentylenetetrazol (PTZ)-induced seizures by stimulating EP1 and EP3 receptors. However, up to the present moment, no study has investigated whether EP1 and EP3 receptors blocking attenuate seizures induced by convulsants other than PTZ. It is also unknown whether Na+,K+-ATPase activity alterations are involved in such an effect. Therefore, in the current study we investigated whether EP1 and EP3 ligands (agonists and antagonists) modulate PTZ- and kainic acid (KA)-induced seizures, and whether alterations in Na+,K+-ATPase activity mediate such a protective effect, in mice. EP1 and EP3 antagonists (ONO-8713 and ONO-AE3-240, respectively, 10 Og/kg, s.c.) attenuated PTZ (60 mg/kg, i.p.)- and KA (20 mg/kg, i.p.)-induced seizures. The respective agonists (ONO-DI-004 and ONO-AE-248, 10 Og/kg, s.c.) facilitated seizures in both acute models, and at noneffective doses, prevented the protective effects of the antagonists. Animals injected with PTZ presented decreased Na+,K+-ATPase activity in the cerebral cortex and hippocampus. On the other hand, animals injected with KA presented increased Na+,K+-ATPase activity in the same cerebral structures at the end of the experiment. These divergent findings suggest that alterations in Na+,K+-ATPase activity in both acute models depends on the convulsant agent used and make difficult to establish a relationship between Na+,K+-ATPase activity and seizure development. Moreover, EP1 and EP3 antagonists administration abolished Na+,K+- ATPase activity alterations induced by PTZ and KA, in such a way that these alterations seem to be related more to the presence of ictal phenomenon itself than to the seizure induction mechanisms. Notwithstanding, the currrent results clearly show that EP1 and EP3 receptors might constitute novel targets for anticonvulsants development, since EP1 and EP3 decreased seizures, regardless of the convulsant agent used. / A epilepsia é uma das disfunções neurológicas mais comuns. Tem sido sugerido que as crises epilépticas podem ser facilitadas pela ocorrência de inflamação. A PGE2 é um dos mediadores inflamatórios mais importantes que, agindo por meio dos receptores EP1 e EP3, facilita as convulsões induzidas por pentilenotetrazol (PTZ). Contudo, até a presente data, nenhum estudo investigou, de maneira sistêmica, se a ativação ou bloqueio de receptores EP1 e EP3 facilitam as convulsões induzidas por outros agentes; tampouco se alterações na atividade da Na+,K+-ATPase estão envolvidas nesse efeito. Assim, no presente estudo, investigamos se ligantes (agonistas e antagonistas) de receptores EP1 e EP3 modificam as crises induzidas por PTZ e ácido caínico (KA), e se tais efeitos estão associados a alterações na atividade da enzima Na+,K+-ATPase, em camundongos. Os antagonistas EP1 e EP3 (ONO-8713 e ONO-AE3-240, respectivamente, 10 Og/Kg, s.c.) atenuaram as convulsões induzidas por PTZ (60 mg/Kg, i.p.) e KA (20 mg/Kg). Os seus respectivos agonistas (ONO-DI-004 e ONO-AE-248 de 10 Og/Kg, s.c.) facilitaram as convulsões em ambos modelos agudos de crises epilépticas e, em doses não efetivas para gerar crises, preveniram os efeitos dos antagonistas. Os animais submetidos à administração de PTZ apresentaram, ao final do experimento, a atividade Na+,K+-ATPásica diminuída no córtex cerebral e hipocampo. Por outro lado, animais tratados com KA apresentaram um aumento na atividade Na+,K+-ATPásica nestas mesmas estruturas, que se correlacionou positivamente com a vigência de status epilepticus no momento do sacrifício. Os achados divergentes no que diz respeito à alteração da atividade da Na+,K+-ATPase nos dois modelos de crises agudas sugere que tais alterações estejam relacionadas ao tipo de agente convulsivante utilizado, e dificultam estabelecer, de forma inequívoca, uma relação entre atividade desta ATPase e sensibilidade à crises agudas. Ademais, a administração de antagonistas EP1 e EP3 aboliu as alterações da atividade da Na+,K+-ATPase induzidas tanto por PTZ como por KA, de tal forma que estas parecem estar mais associadas com o fenômeno ictal em si, do que com os mecanismos de indução da crise. Contudo, os resultados mostram de forma clara que os receptores EP1 e EP3 podem se constituir possíveis novos alvos para o desenvolvimento de drogas antiepilépticas, pois antagonistas EP1 e EP3 diminuíram as crises, independente do agente convulsivante utilizado.

Kainic acid induced excitotoxicity causes pyramidal cell death in the CA3a/b region of the hippocampus. Electrical synapses, gap junctional communication, and single membrane channels in non-junctional membranes (hemichannels) composed of connexin36 (Cx36) have been implicated in both seizure propagation and the spread of excitotoxic cell death. In rats, Cx36 protein is expressed by pyramidal neurons. Localization of protein in mouse, however, is highly controversial. Expression is reported to be restricted to hippocampal interneurons yet the same excitotoxic mechanisms (electrical and metabolic coupling between pyramidal neurons) are invoked to explain the role of Cx36 in excitotoxic pyramidal loss in murine brain. To address this controversy, I show by confocal immunofluorescence and in situ hybridization that Cx36 protein expression is restricted to interneurons and microglia in murine hippocampus and is not expressed by, or is below level of detection in pyramidal neurons. Using behavioural and electrophysiological measures, seizure propagation was found to be moderately enhanced in the absence of Cx36 likely due to the loss of interneuron-mediated synchronous inhibition of the pyramidal cells. Further, CA3a/b neurons die post kainic acid injury in the presence of Cx36 but are protected in Cx36-/- mice. When delayed excitotoxic cell death is maximal, Cx36 is primarily expressed by activated microglia as demonstrated by confocal immunofluorescence, in situ hybridization, and Western blotting. These activated microglia are located in the direct vicinity of, and surrounding cells in the damaged Ca3a/b region. Finally, I show that loss of Cx36 from activated microglia in mice is sufficient to prevent excitotoxic cell death in the CA3a/b with surviving neurons functional as assessed by both electrophysiological and behavioural measures. Together, these data identify a new mechanism of excitotoxic injury, mediated by neuronal-glial interactions, and dependent on microglial Cx36 expression.

Intrastriatal injections of L-Pyroglutamate (L-PGA) in mice produced behavioral and neuropathological effects that resemble in part the kainate-injected rat striatal model of Huntington's Disease (HD). The behavioral responses induced after unilateral injections of L-PGA included circling, postural asymmetry of head and trunk and possible dyskinesias. The neuropil in the injected striatum contained dilated profiles, degenerating neurons and oligodendroglia, and numerous phagocytic microglial-like cells. A dose response relation existed. The size of the lesion (expressed as a percent volume of the striatum destroyed) ranged from 1±0.18% at 0.02 μmoles to 20.2±3.97% at 200 μmoles L-PGA (pH=7.3). L-PGA is a weak neurotoxin when compared to kainic acid. Several factors raise interest in the possible role of L-PGA in HD, including the recently reported elevated plasma levels of L-PGA in some HD patients [51,52], and these are considered in the discussion.

No / Phosphorylation or SUMOylation of the kainate receptor (KAR) subunit GluK2 have both individually been shown to regulate KAR surface expression. However, it is unknown whether phosphorylation and SUMOylation of GluK2 are important for activity-dependent KAR synaptic plasticity. We found that protein kinase C-mediated phosphorylation of GluK2 at serine 868 promotes GluK2 SUMOylation at lysine 886 and that both of these events are necessary for the internalization of GluK2-containing KARs that occurs during long-term depression of KAR-mediated synaptic transmission at rat hippocampal mossy fiber synapses. Conversely, phosphorylation of GluK2 at serine 868 in the absence of SUMOylation led to an increase in KAR surface expression by facilitating receptor recycling between endosomal compartments and the plasma membrane. Our results suggest a role for the dynamic control of synaptic SUMOylation in the regulation of KAR synaptic transmission and plasticity.

No / The surface expression and regulated endocytosis of kainate (KA) receptors (KARs) plays a critical role in neuronal function. PKC can modulate KAR trafficking, but the sites of action and molecular consequences have not been fully characterized. Small ubiquitin-like modifier (SUMO) modification of the KAR subunit GluK2 mediates agonist-evoked internalization, but how KAR activation leads to GluK2 SUMOylation is unclear. Here we show that KA stimulation causes rapid phosphorylation of GluK2 by PKC, and that PKC activation increases GluK2 SUMOylation both in vitro and in neurons. The intracellular C-terminal domain of GluK2 contains two predicted PKC phosphorylation sites, S846 and S868, both of which are phosphorylated in response to KA. Phosphomimetic mutagenesis of S868 increased GluK2 SUMOylation, and mutation of S868 to a nonphosphorylatable alanine prevented KA-induced SUMOylation and endocytosis in neurons. Infusion of SUMO-1 dramatically reduced KAR-mediated currents in HEK293 cells expressing WT GluK2 or nonphosphorylatable S846A mutant, but had no effect on currents mediated by the S868A mutant. These data demonstrate that agonist activation of GluK2 promotes PKC-dependent phosphorylation of S846 and S868, but that only S868 phosphorylation is required to enhance GluK2 SUMOylation and promote endocytosis. Thus, direct phosphorylation by PKC and GluK2 SUMOylation are intimately linked in regulating the surface expression and function of GluK2-containing KARs.

L'épilepsie du lobe temporal (ELT) est la forme la plus fréquente de l'épilepsie chez l'adulte. Elle se traduit par des crises spontanées et récurrentes, qui sont résistantes à tout traitement dans 90% des cas. Une agression initiale du cerveau (traumatisme crânien, méningite, convulsions fébriles etc.), est souvent à l'origine de la transformation d'un cerveau « sain » en cerveau épileptique. L'ensemble des processus responsables de cette transition s'appelle l'épileptogenèse. Pouvoir bloquer et/ou retarder l'épileptogenèse chez les patients à risque est une question de santé majeure. En plus des crises, l'ELT soulève d'autres questions. Elle est souvent associée à des déficits cognitifs, qui sont la conséquence de la réorganisation des circuits neuronaux. Ces déficits pourraient être traités de façon indépendante de l'épilepsie elle-même. Le projet de recherche de cette thèse s'inscrit dans ce cadre général. / Temporal Lobe Epilepsy (TLE) is the most common form of epilepsy in adults. It translates into spontaneous and recurrent seizures, which are resistant to any treatment in 90% of cases. An initial brain insult (head injury, meningitis, febrile seizures etc.), is often the cause of the transformation of a "healthy" brain into an epileptic one. The process responsible for this transition is called epileptogenesis. Blocking and/or delaying epileptogenesis in at-risk patients is a key issue for public health. In addition to the seizures, TLE raises other problems. It is often associated with cognitive deficits, which are the result of the reorganization of neuronal circuits. These deficits may be treated independently of epilepsy itself. The work presented here fits into this general framework.

Les épilepsies acquises se déclarent après un processus graduel appelé épileptogenèse. Bien que cliniquement silencieux, ce processus implique des modifications fonctionnelles observables notamment par électroencéphalographie. Cette thèse vise i) à identifier des marqueurs électrophysiologiques apparaissant au cours de l’épileptogenèse, et ii) à comprendre les modifications physiopathologiques sous-jacentes responsables de ces marqueurs et de leur évolution temporelle. Dans un premier temps, nous avons, dans un modèle d’épilepsie partielle chez la souris, monitoré des signaux électrophysiologiques intracérébraux pendant la mise en place de la maladie. Nous avons observé dans ces signaux expérimentaux, l’émergence d’événements transitoires pathologiques appelés pointes épileptiques. Nous avons développé des méthodes de traitement du signal pour détecter et caractériser automatiquement ces événements. Ainsi, nous avons pu mettre en évidence certains changements dans la forme des pointes épileptiques au cours de l’épileptogenèse ; en particulier l’apparition et l’augmentation d’une onde qui suit la pointe épileptique. Une hypothèse défendue dans ces travaux est que ces changements morphologiques peuvent constituer des marqueurs de l’épileptogenèse dans ce modèle animal. Dans un second temps, afin d’interpréter ces modifications électrophysiologiques en termes de processus neurophysiologiques sous-jacents, nous avons implémenté un modèle biomathématique, physiologiquement argumenté, capable de simuler des pointes épileptiques. Formellement, ce modèle est un système dynamique non linéaire qui reproduit les interactions synaptiques (excitatrices et inhibitrices) dans une population de neurones. Une analyse de sensibilité de ce modèle a permis de mettre en évidence le rôle critique de certains paramètres de connectivité dans la morphologie des pointes. Nos résultats montrent en effet, qu’une diminution de l’inhibition GABAergique entraîne un accroissement de l’onde dans les pointes épileptiques. À partir du modèle théorique, nous avons pu ainsi émettre des hypothèses sur les modifications opérant au cours du processus d’épileptogenèse. Ces hypothèses ont pu être en partie vérifiées expérimentalement en bloquant artificiellement l’inhibition GABAergique, dans le modèle in vivo chez la souris, et dans un modèle in vitro chez le rat. En conclusion, ce travail de thèse fournit, dans un modèle animal, un biomarqueur électrophysiologique de l’épileptogenèse et tente d’expliquer, grâce à une modélisation biomathématique, les processus neurophysiologiques sous-jacents qu’il reflète. / Acquired epilepsies occur after a process called epileptogenesis. Although clinically silent, this process involves some functional modifications which can be observed by electroencephalography. The objectives of this thesis are i) to identify electrophysiological markers occurring during epileptogenesis, and ii) to understand which underlying pathophysiological modifications are responsible for these markers and their evolution. Firstly, using an in vivo experimental mouse model of partial epilepsy, we have monitored intracranial electrophysiological signals during epileptogenesis. We observed the emergence of pathological transient events called epileptic spikes. We have developed signal processing methods in order to automatically detect and characterize these events. Hence, we observed and quantified morphological changes of epileptic spikes during epileptogenesis. In particular, we noticed the emergence and the increase of a wave which directly follows the spike component. In this work, we defend the hypothesis that these morphological modifications can constitute markers of the epileptogenesis process in this animal model of epilepsy. Secondly, in order to interpret these electrophysiological modifications in terms of underlying pathophysiological processes, we have implemented a computational model able to simulate epileptic spikes. This neural mass model is a neurophysiologically-plausible mesoscopic representation of synaptic interactions (excitation and inhibition) in the hippocampus. Based on a sensitivity analysis of model parameters, we were able to determine some connectivity parameters that play a key role in the morphology of simulated epileptic spikes. In particular, our results show that a diminution of GABAergic inhibition leads to an increase of the aforementioned wave. Thus, using this theoretical model, we defined some hypotheses about pathophysiological modifications occurring during the epileptogenesis process. One of these hypotheses has been confirmed in blocking GABAa receptors in the in vivo mouse model, as well as in an in vitro model (rat, organotypic slices). In summary, based on the shape features of epileptic spikes, we devised an electrophysiological biomarker of epileptogenesis observed in a mouse model but useful in Human studies as well. Moreover, a computational modeling approach has permitted to suggest which pathophysiological processes might underlie this biomarker.

There has been an awareness of nerve poisons from ancient times. At the dawn of the twentieth century, the actions and mechanisms of these poisons were uncovered by modern physiological and biochemical experimentation. However, the era of selective neurotoxins began with the pioneering studies of R. Levi-Montalcini through her studies of the neurotrophin "nerve growth factor" (NGF), a protein promoting growth and development of sensory and sympathetic noradrenergic nerves. An antibody to NGF, namely, anti-NGF - developed in the 1950s in a collaboration with S. Cohen - was shown to produce an "immunosympathectomy" and virtual lifelong sympathetic denervation. These Nobel Laureates thus developed and characterized the first identifiable selective neurotoxin. Other selective neurotoxins were soon discovered, and the compendium of selective neurotoxins continues to grow, so that today there are numerous selective neurotoxins, with the potential to destroy or produce dysfunction of a variety of phenotypic nerves. Selective neurotoxins are of value because of their ability to selectively destroy or disable a common group of nerves possessing (1) a particular neural transporter, (2) a unique set of enzymes or vesicular transporter, (3) a specific type of receptor or (4) membranous protein, or (5) other uniqueness. The era of selective neurotoxins has developed to such an extent that the very definition of a "selective" neurotoxin has warped. For example, (1) N-methyl-D- aspartate receptor (NMDA-R) antagonists, considered to be neuroprotectants by virtue of their prevention of excitotoxicity from glutamate receptor agonists, actually lead to the demise of populations of neurons with NMDA receptors, when administered during ontogenetic development. The mere lack of natural excitation of this nerve population, consequent to NMDA-R block, sends a message that these nerves are redundant - and an apoptotic cascade is set in motion to eliminate these nerves. (2) The rodenticide rotenone, a global cytotoxin that acts mainly to inhibit complex I in the respiratory transport chain, is now used in low dose over a period of weeks to months to produce relatively selective destruction of substantia nigra dopaminergic nerves and promote alpha-synuclein deposition in brain to thus model Parkinson's disease. Similarly, (3) glial toxins, affecting oligodendrocytes or other satellite cells, can lead to the damage or dysfunction of identifiable groups of neurons. Consequently, these toxins might also be considered as "selective neurotoxins," despite the fact that the targeted cell is nonneuronal. Likewise, (4) the dopamine D2-receptor agonist quinpirole, administered daily for a week or more, leads to development of D2-receptor supersensitivity - exaggerated responses to the D2-receptor agonist, an effect persisting lifelong. Thus, neuroprotectants can become "selective" neurotoxins; nonspecific cytotoxins can become classified as "selective" neurotoxins; and receptor agonists, under defined dosing conditions, can supersensitize and thus be classified as "selective" neurotoxins. More examples will be uncovered as the area of selective neurotoxins expands. The description and characterization of selective neurotoxins, with unmasking of their mechanisms of action, have led to a level of understanding of neuronal activity and reactivity that could not be understood by conventional physiological observations. This chapter will be useful as an introduction to the scope of the field of selective neurotoxins and provide insight for in-depth analysis in later chapters with full descriptions of selective neurotoxins.

The alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR) subunit GluR2, which regulates excitotoxicity and the inflammatory cytokine tumour necrosis factor alpha (TNFalpha) have both been implicated in motor neurone vulnerability in amyotrophic lateral sclerosis/motor neurone disease. TNFalpha has been reported to increase cell surface expression of AMPAR subunits to increase synaptic strength and enhance excitotoxicity, but whether this mechanism occurs in motor neurones is unknown. We used primary cultures of mouse motor neurones and cortical neurones to examine the interaction between TNFalpha receptor activation, GluR2 availability, AMPAR-mediated calcium entry and susceptibility to excitotoxicity. Short exposure to a physiologically relevant concentration of TNFalpha (10 ng/mL, 15 min) caused a marked redistribution of both GluR1 and GluR2 to the cell surface as determined by cell surface biotinylation and immunofluorescence. Using fura-2-acetoxymethyl ester microfluorimetry, we showed that exposure to TNFalpha caused a rapid reduction in the peak amplitude of AMPA-mediated calcium entry in a PI3-kinase and p38 kinase-dependent manner, consistent with increased insertion of GluR2-containing AMPAR into the plasma membrane. This resulted in a protection of motor neurones against kainate-induced cell death. Our data therefore, suggest that TNFalpha acts primarily as a physiological regulator of synaptic activity in motor neurones rather than a pathological drive in amyotrophic lateral sclerosis.