Multimodal Characterization of Seizures in Zebrafish Larvae

Abstract

Epilepsy accounts for a significant proportion of the world’s disease burden. Indeed, many research efforts are produced both to investigate the basic mechanism ruling its genesis and to find more effective therapies. In this framework, the use of zebrafish larvae, owing to their peculiar features, offers a great opportunity. Here, we employ transgenic zebrafish larvae expressing GCaMP6s in all neurons to characterize functional alterations occurring during seizures induced by pentylenetetrazole. Using a custom two-photon light-sheet microscope, we perform fast volumetric functional imaging of the entire larval brain, investigating how different brain regions contribute to seizure onset and propagation. Moreover, employing a custom behavioral tracking system, we outline the progressive alteration of larval swim kinematics, resulting from different grades of seizures. Collectively, our results show that the epileptic larval brain undergoes transitions between diverse neuronal activity regimes. Moreover, we observe that different brain regions are progressively recruited into the generation of seizures of diverse severity. We demonstrate that midbrain regions exhibit highest susceptibility to the convulsant effects and that, during periods preceding abrupt hypersynchronous paroxysmal activity, they show a consistent increase in functional connectivity. These aspects, coupled with the hub-like role that these regions exert, represent important cues in their identification as epileptogenic hubs.

Two-photon all-optical electrophysiology for the dissection of larval zebrafish brain functional connectivity

“…Screened embryos were transferred to a Petri dish containing fresh fish water and kept in an incubator at 28.5 °C until 5 dpf. Zebrafish larvae were mounted as previously described 86 . Briefly, each larva was transferred into a reaction tube containing 1.5% (w/v) low-gelling temperature agarose (A9414, Sigma-Aldrich) in fish water, maintained fluid on a heater set at 38°C.…”

Zebrafish as an Innovative Tool for Epilepsy Modeling: State of the Art and Potential Future Directions

“…In this framework, different approaches ranging from the single patched cell to an electroencephalogram of the entire brain have been developed, used, and optimized. The currently available in vivo technologies to observe and monitor the electrographic seizure events in ZF with chemically-induced seizures or in ZF epilepsy models (epileptic morphant/transgenic fish) consist generally of video tracking approaches to assess changes in the neurobehavioral profile of ZF (swimming activities, spontaneous movements, and touch response), as already described [31,86], as well as sophisticated and invasive tools [31,112,113], microfluidic systems [114,115], and different fluorescence microscopy techniques to monitor the electrographic events [116][117][118].…”

“…First, the imaging approaches are not invasive; moreover, the microscopy measurements provide a recording of the activity in thousand neurons or the whole brain [118], allowing for the getting of information from the single-cell behavior up to the whole-brain dynamics. Transgenic ZF with calcium indicator has been employed to monitor the neuronal activity or perform in vivo imaging of network events or fast volumetric imaging using different fluorescence techniques including simple fluorescence microscopy, fast confocal microscopy, two-photon light-sheet microscopy and spinning disk confocal microscopy [116][117][118]125,126].…”

Developing Novel Experimental Models of m-TORopathic Epilepsy and Related Neuropathologies: Translational Insights from Zebrafish

“…Another highly promising method for studying functional activity of zebrafish brain during epilepsy is light sheet microscopic imaging, based on layer-by-layer CNS photography to assess region-specific activity of larval fish [ 103 ]. For example, Ca 2+ imaging can be used to visualize and record real-time epileptiform activity in zebrafish brain and identify neural circuits involved in epilepsy [ 104 ].…”