Microglia Mitigate Neuronal Activation in a Zebrafish Model of Dravet Syndrome.

It has been known for a long time that epileptic seizures provoke brain neuroinflammation involving the activation of microglial cells. However, the role of these cells in this disease context and the consequences of their inflammatory activation on subsequent neuron network activity remain poorly understood so far. To fill this gap of knowledge and gain a better understanding of the role of microglia in the pathophysiology of epilepsy, we used an established zebrafish Dravet syndrome epilepsy model based on Scn1Lab sodium channel loss-of-function, combined with live microglia and neuronal Ca2+ imaging, local field potential (LFP) recording, and genetic microglia ablation. Data showed that microglial cells in scn1Lab-deficient larvae experiencing epileptiform seizures displayed morphological and biochemical changes characteristic of M1-like pro-inflammatory activation; i.e., reduced branching, amoeboid-like morphology, and marked increase in the number of microglia expressing pro-inflammatory cytokine Il1ß. More importantly, LFP recording, Ca2+ imaging, and swimming behavior analysis showed that microglia-depleted scn1Lab-KD larvae displayed an increase in epileptiform seizure-like neuron activation when compared to that seen in scn1Lab-KD individuals with microglia. These findings strongly suggest that despite microglia activation and the synthesis of pro-inflammatory cytokines, these cells provide neuroprotective activities to epileptic neuronal networks, making these cells a promising therapeutic target in epilepsy.

Bixafen, a succinate dehydrogenase inhibitor fungicide, causes microcephaly and motor neuron axon defects during development.

Succinate dehydrogenase inhibitors (SDHIs), the most widely used fungicides in agriculture today, act by blocking succinate dehydrogenase (SDH), an essential and evolutionarily conserved component of mitochondrial respiratory chain. Recent results showed that several SDHIs used as fungicides not only inhibit the SDH activity of target fungi but also block this activity in human cells in in vitro models, revealing a lack of specificity and thus a possible health risk for exposed organisms, including humans. Despite the frequent detection of SDHIs in the environment and on harvested products and their increasing use in modern agriculture, their potential toxic effects in vivo, especially on neurodevelopment, are still under-evaluated. Here we assessed the neurotoxicity of bixafen, one of the latest-generation SDHIs, which had never been tested during neurodevelopment. For this purpose, we used a well-known vertebrate model for toxicity testing, namely zebrafish transparent embryos, and live imaging using transgenic lines labelling the brain and spinal cord. Here we show that bixafen causes microcephaly and defects on motor neuron axon outgrowth and their branching during development. Our findings show that the central nervous system is highly sensitive to bixafen, thus demonstrating in vivo that bixafen is neurotoxic in vertebrates and causes neurodevelopmental defects. This work adds to our knowledge of the toxic effect of SDHIs on neurodevelopment and may help us take appropriate precautions to ensure protection against the neurotoxicity of these substances.

Organophosphorus diisopropylfluorophosphate (DFP) intoxication in zebrafish larvae causes behavioral defects, neuronal hyperexcitation and neuronal death.

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 respiratory distress, hypertension, and neuron damage that may lead to epileptic seizures and permanent cognitive 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.

A Fast, Simple, and Affordable Technique to Measure Oxygen Consumption in Living Zebrafish Embryos.

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.

Neurons Expressing Pathological Tau Protein Trigger Dramatic Changes in Microglial Morphology and Dynamics.

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.

Defective Excitatory/Inhibitory Synaptic Balance and Increased Neuron Apoptosis in a Zebrafish Model of Dravet Syndrome.

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.