Mapping neuronal activity during the onset and propagation of epileptic seizures can provide a better understanding of the mechanisms underlying this pathology and improve our approaches to the development of new drugs. Recently, zebrafish has become an important model for studying epilepsy both in basic research and in drug discovery. Here, we employed a transgenic line with pan-neuronal expression of the genetically-encoded calcium indicator GCaMP6s to measure neuronal activity in zebrafish larvae during seizures induced by pentylenetretrazole (PTZ). With this approach, we mapped neuronal activity in different areas of the larval brain, demonstrating the high sensitivity of this method to different levels of alteration, as induced by increasing PTZ concentrations, and the rescuing effect of an anti-epileptic drug. We also present simultaneous measurements of brain and locomotor activity, as well as a high-throughput assay, demonstrating that GCaMP measurements can complement behavioural assays for the detection of subclinical epileptic seizures, thus enabling future investigations on human hypomorphic mutations and more effective drug screening methods. Notably, the methodology described here can be easily applied to the study of many human neuropathologies modelled in zebrafish, allowing a simple and yet detailed investigation of brain activity alterations associated with the pathological phenotype.

Light-sheet microscopy (LSM), in combination with intrinsically transparent zebrafish larvae, is a method of choice to observe brain function with high frame rates at cellular resolution. Inherently to LSM, however, residual opaque objects cause stripe artifacts, which obscure features of interest and, during functional imaging, modulate fluorescence variations related to neuronal activity. Here, we report how Bessel beams reduce streaking artifacts and produce high-fidelity quantitative data demonstrating a fivefold increase in sensitivity to calcium transients and a 20-fold increase in accuracy in the detection of activity correlations in functional imaging. Furthermore, using principal component analysis, we show that measurements obtained with Bessel beams are clean enough to reveal in one-shot experiments correlations that can not be averaged over trials after stimuli as is the case when studying spontaneous activity. Our results not only demonstrate the contamination of data by systematic and random errors through conventional Gaussian illumination and but,furthermore, quantify the increase in fidelity of such data when using Bessel beams.

We employed this system to investigate, in real-time on a brain-wide scale, the onset and propagation of acute seizures, as induced by the convulsant drug pentylenetetrazol (PTZ), avoiding detrimental visual stimulation on a highly susceptible system such as an epileptic brain. At a moderate PTZ concentration we observed a widespread increase in nervous activity synchronization with the progression of the exposure time to the drug, particularly in the optic tectum, associated with an unexpected synchronization decrease happening in small spatially-defined areas. At saturating PTZ concentrations we instead observed a brain-wide functional connectivity reorganization and the emergence of distinctive phases of ictal and postictal activity. During the ictal phase the degree of synchronization in the neuronal activity dramatically increases in the whole-brain. On the contrary, the degree of neuronal synchrony in the postictal phase resembles the control condition, with the exception of the dorsal thalamus, where it increases, and subregions of the spinal cord, where it unexpectedly decreases.The volumetric frame rate of our system allowed us to observe the emergence of previously unreported fast rhythmic ictal waves propagating in postero-anterior direction, which we termed caudo-rostral ictal waves (CRIWs), spanning across the whole larval encephalon in about 1 s during the ictal phase.In conclusion, the presented 2P LSF microscope design affords high spatio-temporal resolution while avoiding visual stimuli and allows unprecedented access to whole zebrafish brain epileptic dynamics.

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