Dravet syndrome is a type of severe childhood epilepsy that responds poorly to current anti-epileptic drugs. In recent years, zebrafish disease models with Scn1Lab sodium channel deficiency have been generated to seek novel anti-epileptic drug candidates, some of which are currently undergoing clinical trials. However, the spectrum of neuronal deficits observed following Scn1Lab depletion in zebrafish larvae has not yet been fully explored. To fill this gap and gain a better understanding of the mechanisms underlying neuron hyperexcitation in Scn1Lab-depleted larvae, we analyzed neuron activity in vivo using combined local field potential recording and transient calcium uptake imaging, studied the distribution of excitatory and inhibitory synapses and neurons as well as investigated neuron apoptosis. We found that Scn1Lab-depleted larvae displayed recurrent epileptiform seizure events, associating massive synchronous calcium uptakes and ictal-like local field potential bursts. Scn1Lab-depletion also caused a dramatic shift in the neuronal and synaptic balance toward excitation and increased neuronal death. Our results thus provide in vivo evidence suggesting that Scn1Lab loss of function causes neuron hyperexcitation as the result of disturbed synaptic balance and increased neuronal apoptosis.

Microglial cells, the resident macrophages of the brain, are important players in the pathological process of numerous neurodegenerative disorders, including tauopathies, a heterogeneous class of diseases characterized by intraneuronal Tau aggregates. However, microglia response in Tau pathologies remains poorly understood. Here, we exploit a genetic zebrafish model of tauopathy, combined with live microglia imaging, to investigate the behavior of microglia in vivo in the disease context. Results show that while microglia were almost immobile and displayed long and highly dynamic branches in a wild-type context, in presence of diseased neurons, cells became highly mobile and displayed morphological changes, with highly mobile cell bodies together with fewer and shorter processes. We also imaged, for the first time to our knowledge, the phagocytosis of apoptotic tauopathic neurons by microglia in vivo and observed that microglia engulfed about as twice materials as in controls. Finally, genetic ablation of microglia in zebrafish tauopathy model significantly increased Tau hyperphosphorylation, suggesting that microglia provide neuroprotection to diseased neurons. Our findings demonstrate for the first time the dynamics of microglia in contact with tauopathic neurons in vivo and open perspectives for the real-time study of microglia in many neuronal diseases.

Dravet syndrome is a type of severe childhood epilepsy that responds poorly to current anti-epileptic drugs. In recent years, zebrafish disease models with Scn1Lab sodium channel deficiency have been generated to seek novel anti-epileptic drug candidates, some of which are currently undergoing clinical trials. However, the spectrum of neuronal deficits observed following Scn1Lab depletion in zebrafish larvae has not yet been fully explored. To fill this gap and gain a better understanding of the mechanisms underlying neuron hyperexcitation in Scn1Lab-depleted larvae, we analyzed neuron activity in vivo using combined local field potential recording and transient calcium uptake imaging, studied the distribution of excitatory and inhibitory synapses and neurons as well as investigated neuron apoptosis. We found that Scn1Lab-depleted larvae displayed recurrent epileptiform seizure events, associating massive synchronous calcium uptakes and ictal-like local field potential bursts. Scn1Lab-depletion also caused a dramatic shift in the neuronal and synaptic balance toward excitation and increased neuronal death. Our results thus provide in vivo evidence suggesting that Scn1Lab loss of function causes neuron hyperexcitation as the result of disturbed synaptic balance and increased neuronal apoptosis.

In all animal species, oxygen consumption is a key process that is partially impaired in a large number of pathological situations and thus provides informative details on the physiopathology of the disease. In this study, we describe a simple and affordable method to precisely measure oxygen consumption in living zebrafish larvae using a spectrofluorometer and the MitoXpress Xtra Oxygen Consumption Assay. In addition, we used zebrafish larvae treated with mitochondrial respiratory chain inhibitors, antimycin A or rotenone, to verify that our method enables precise and reliable measurements of oxygen consumption.

With millions of intoxications each year and over 200,000 deaths, organophosphorus (OP) compounds are an important public health issue worldwide. OP poisoning induces cholinergic syndrome, with muscle weakness, hypertension, and neuron damage that may lead to epileptic seizures and permanent psychomotor deficits. Existing countermeasures are lifesaving but do not prevent long-lasting neuronal comorbidities, emphasizing the urgent need for animal models to better understand OP neurotoxicity and identify novel antidotes. Here, using diisopropylfluorophosphate (DFP), a prototypic and moderately toxic OP, combined with zebrafish larvae, we first showed that DFP poisoning caused major acetylcholinesterase inhibition, resulting in paralysis and CNS neuron hyperactivation, as indicated by increased neuronal calcium transients and overexpression of the immediate early genes fosab, junBa, npas4b , and atf3 . In addition to these epileptiform seizure-like events, DFP-exposed larvae showed increased neuronal apoptosis, which were both partially alleviated by diazepam treatment, suggesting a causal link between neuronal hyperexcitation and cell death. Last, DFP poisoning induced an altered balance of glutamatergic/GABAergic synaptic activity with increased NR2B-NMDA receptor accumulation combined with decreased GAD65/67 and gephyrin protein accumulation. The zebrafish DFP model presented here thus provides important novel insights into the pathophysiology of OP intoxication, making it a promising model to identify novel antidotes.

L'épilepsie est une maladie neurologique qui touche plus de 50 millions de personnes dans le monde. Elle se caractérise par des crises récurrentes dues à la surexcitation synchrone et spontanée de populations neuronales du cerveau. Les crises sont de nature très variable et les symptômes dépendent de la zone du cerveau touchée et de son étendue. Le terme «troubles épileptiques» est par conséquent préféré. Ceux-ci peuvent avoir de nombreuses causes, soient génétiques (par exemple, le syndrome de Dravet, une épilepsie infantile rare, provoquée dans 80% des cas par la mutation hétérozygote du gène SCN1A), soient environnementales (par exemple, après un empoisonnement aux organophosphorés, des composés présents dans les pesticides et les agents de guerre neurotoxiques). Dans les deux cas, les traitements actuels ne permettent pas un contrôle optimal des crises. Une meilleure compréhension de la physiopathologie de ces différentes formes d'épilepsie est donc nécessaire pour trouver de nouvelles cibles thérapeutiques et de nouveaux anticonvulsivants. Les cellules microgliales, les macrophages résidents du cerveau ont de nombreuses fonctions qui varient en fonction de la maturité du cerveau. Les microglies sont les gardiennes de l'homéostasie cérébrale, assurant en permanence le bon fonctionnement des neurones. Ce sont des cellules immunitaires capables de moduler leur activité en fonction des dangers qu'elles détectent. De plus, elles ont un rôle particulier dans la plasticité synaptique et la modulation de l'excitabilité neuronale. Ces différents rôles ont suscité de nombreuses hypothèses sur l'implication de ces cellules dans la physiopathologie des troubles épileptiques. Pour certaines, les microglies sont nocives pour l'excitabilité des neurones, par leur activation et la sécrétion chronique de cytokines pro-inflammatoires. Pour d'autres, elles ont un rôle bénéfique, la microglie tamponnant l'hyperexcitabilité neuronale et diminuant ainsi la fréquence des crises. L'objectif de mon travail de thèse était d'étudier les mécanismes de l'épileptogenèse impliquant les cellules microgliales afin d'identifier de nouvelles cibles thérapeutiques. J'ai développé deux modèles d'épilepsie chez le poisson zèbre, un modèle génétique du syndrome de Dravet et un modèle d'empoisonnement aux organophosphorés. Ceux-ci m'ont permis d'étudier les modifications du système nerveux central au cours de l'épileptogenèse. J'ai ainsi montré un déséquilibre de la balance excitateur/inhibiteur vers l'excitation qui pourrait déclencher des crises d'épilepsie. En utilisant le modèle de Dravet, j'ai également caractérisé les changements morphologiques, comportementaux et moléculaires des cellules microgliales après des crises. Ces travaux améliorent notre compréhension des conséquences des crises d'épilepsie dans le cerveau et contribuent à ouvrir la voie à la découverte de nouvelles cibles thérapeutiques pour traiter différentes formes d'épilepsie.