Defective excitatory/inhibitory synaptic balance and increased neuron apoptosis in a zebrafish model of Dravet syndrome

Abstract

Dravet syndrome is a type of severe childhood epilepsy that responds poorly to current antiepileptic 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.

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

“…In humans suffering from acute OP intoxication, whenever neuronal hyperexcitation is not rapidly treated, epileptic seizures eventually develop into SE, a major life-threatening neurological condition 6,45 , which also causes long-term brain damage 46,47 . Interestingly, in the zebrafish model of OP poisoning presented here, we observed that almost all the larvae displayed numerous massive calcium transients from 2 to 3 h after DFP exposure onward, which were strongly reminiscent of those associated with generalized seizures seen in both genetic and pharmacological zebrafish epilepsy models 14,26 . Altogether, these findings strongly suggest that DFP exposure causes epileptiform neuronal hyperexcitation in zebrafish larvae, eventually leading to a status epilepticus-like condition.…”

“…Five dpf larvae from the transgenic line Tg[Huc:GCaMP5G] were treated with DFP or vehicle (DMSO) and calcium transients were recorded during the following 6 h using time-lapse confocal microscopy ( Supplementary Videos 1 and 2). Interestingly, in DFP-treated larvae, we observed numerous intense calcium transients, from approximately 1 h post-exposure onward, which were highly reminiscent of those seen in zebrafish genetic epilepsy models 14 . Moreover, these calcium transients were mostly seen in the neuropil region, where a large number of synaptic connections occur ( Fig.…”

“…Using qRT-PCR analysis of brain RNAs, we next showed that DFP exposure also induced a massive accumulation of transcripts of the IEGs fosab, atf3, junBa, and npas4b, providing another indication that DFP induces a massive neuronal hyperexcitation in zebrafish larvae. To further investigate whether DFP exposure causes neuronal hyperexcitation, we used calcium imaging, a recent technology that enables the visualization of neuronal excitation and epileptic seizures in living zebrafish larvae 14,26,44 . Interestingly, in larvae exposed to acute DFP poisoning, we observed a massive increase in the number of neuronal calcium transients compared to those seen in controls, and also a significant increase in the amplitude of the transients in DFP-treated larvae.…”

“…We then sought to visualize the increased neuronal activity in larvae exposed to DFP using calcium imaging (Fig. 3a, b), a technique that fully reflects neuronal activity in zebrafish epilepsy models in vivo 14,26 . Five dpf larvae from the transgenic line Tg[Huc:GCaMP5G] were treated with DFP or vehicle (DMSO) and calcium transients were recorded during the following 6 h using time-lapse confocal microscopy ( Supplementary Videos 1 and 2).…”

“…Here, we describe a zebrafish model of OP poisoning and characterize the neuron defects induced by acute intoxication. Over the past decade, besides its rapidly expanding use as a human disease model [12][13][14][15][16] , the zebrafish has become one of the leading animal models for toxicology research and drug discovery 17 . The zebrafish is a versatile, powerful and easy-tobreed vertebrate model that offers significant advantages for in vivo drug screening and neurotoxicology investigations.…”