A "two-hit" pharmacological seizure model in zebrafish for studying microglia dynamics in the developing epileptic brain


Teresa G. Martins, Remon Soliman, Cristina Donato, Maria Lorena Cordero-Maldonado, Corrado Ameli, Laurent Mombaerts, Alexander Skupin, Francesca Peri, Alexander D. Crawford

Epilepsy is a chronic brain disorder characterized by unprovoked and recurrent seizures, of which 60% are of unknown etiology. Recent studies implicate microglia in the pathophysiology of epilepsy. However, their role in this process, e.g. following early-life seizures, remains poorly understood in part due to the lack of suitable experimental models allowing the in vivo imaging of microglial activity using minimally-invasive techniques. Given the advantage of zebrafish larvae for such imaging approaches, we sought to develop a “two-hit” pharmacological seizure model in zebrafish suitable for studying microglia response after acute seizures. Towards this end, different concentrations of kainate were microinjected intravenously into 3 days post-fertilization transgenic zebrafish larvae with mCherry-labeled microglia followed by acute incubation with pentylenetetrazole at 5 days post-fertilization. Kainate-treated larvae exhibited increased numbers of microglia in the brain, and were more susceptible to subsequent pentylenetetrazole-induced seizures, as shown by higher locomotor and encephalographic activity as compared to vehicle-injected larvae. These results are comparable to experimental kainate-induced rodent seizure models and suggest the suitability of our model for future studies. Such studies could contribute to elucidate microglial dynamic changes after seizure induction in the developing brain, and to understand how these modulate seizure susceptibility later in life.

Videos


Videos demonstrating the phenotypes after KA injection.

3 days post-fertilization larvae from the line Tg(csf1r:Gal4)i186; Tg(UAS.nfsB.mCherry)i149 outcrossed to nacw2 after systemic injection with PBS. The video shows that all the larvae display normal behavior and respond to touch (escape response) upon a soft tail touch with a fine pointer.

3 days post-fertilization larvae from the line Tg(csf1r:Gal4)i186; Tg(UAS.nfsB.mCherry)i149 outcrossed to nacw2 after systemic injection with 0.25 mM KA. The video shows that for some larvae only there is occasional mild whole-body trembling and no escape response upon a soft tail touch with a fine pointer.

3 days post-fertilization larvae from the line Tg(csf1r:Gal4)i186; Tg(UAS.nfsB.mCherry)i149 outcrossed to nacw2 after systemic injection with 0.5 mM KA. The video shows that all larvae display occasional whole-body trembling and movement. Additionally they do not respond to touch (escape response) upon a soft tail touch with a fine pointer.

3 days post-fertilization larvae from the line Tg(csf1r:Gal4)i186; Tg(UAS.nfsB.mCherry)i149 outcrossed to nacw2 after systemic injection with 1 mM KA. The video shows that all larvae have occasional whole-body trembling and do not respond to a soft tail touch with a fine pointer.