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.

In recent years, light-sheet fluorescence microscopy (LSFM) has found a broad application for imaging of diverse biological samples, ranging from sub-cellular structures to whole animals, both in-vivo and ex-vivo, owing to its many advantages relative to point-scanning methods. By providing the selective illumination of sample single planes, LSFM achieves an intrinsic optical sectioning and direct 2D image acquisition, with low out-of-focus fluorescence background, sample photo-damage and photo-bleaching. On the other hand, such an illumination scheme is prone to light absorption or scattering effects, which lead to uneven illumination and striping artifacts in the images, oriented along the light sheet propagation direction. Several methods have been developed to address this issue, ranging from fully optical solutions to entirely digital post-processing approaches. In this work, we present them, outlining their advantages, performance and limitations.

Light-sheet fluorescence microscopy (LSFM) enables real-time whole-brain functional imaging in zebrafish larvae. Conventional one-photon LSFM can however induce undesirable visual stimulation due to the use of visible excitation light. The use of two-photon (2P) excitation, employing near-infrared invisible light, provides unbiased investigation of neuronal circuit dynamics. However, due to the low efficiency of the 2P absorption process, the imaging speed of this technique is typically limited by the signal-to-noise-ratio. Here, we describe a 2P LSFM setup designed for non-invasive imaging that enables quintuplicating state-of-the-art volumetric acquisition rate of the larval zebrafish brain (5 Hz) while keeping low the laser intensity on the specimen. We applied our system to the study of pharmacologically-induced acute seizures, characterizing the spatial-temporal dynamics of pathological activity and describing for the first time the appearance of caudo-rostral ictal waves (CRIWs).

Zebrafish has become an essential model organism in modern biomedical research. Owing to its distinctive features and high grade of genomic homology with humans, it is increasingly employed to model diverse neurological disorders, both through genetic and pharmacological intervention. The use of this vertebrate model has recently enhanced research efforts, both in the optical technology and in the bioengineering fields, aiming at developing novel tools for high spatiotemporal resolution imaging. Indeed, the ever-increasing use of imaging methods, often combined with fluorescent reporters or tags, enable a unique chance for translational neuroscience research at different levels, ranging from behavior (whole-organism) to functional aspects (whole-brain) and down to structural features (cellular and subcellular). In this work, we present a review of the imaging approaches employed to investigate pathophysiological mechanisms underlying functional, structural, and behavioral alterations of human neurological diseases modeled in zebrafish.

Fluorescence microscopy, due to its combination of molecular specificity and high contrast, has broad application in a wide range of research areas, from cell biology to neuroscience.^1,2 In particular, light-sheet fluorescence microscopy (LSFM) has become one of the fastest growing techniques for imaging of three-dimensional (3-D) thick samples.^{3 – 5} By illuminating a single plane of the sample, it provides intrinsic optical sectioning and fast image recording, while minimizing out-of-focus fluorescence background and reducing sample photodamage and photobleaching. In such a microscope, the light-sheet is usually created by means of a cylindrical lens³ or by rapidly scanning a Gaussian (or a nondiffracting) beam, as in digital scanned laser light-sheet microscopy (DSLM).^{6 – 8} The induced fluorescence is imaged through a simple wide-field detection path. However, when 3-D imaging is performed in turbid samples, the illumination light can suffer from scattering effects which lead to spurious background signals that cannot be rejected by the wide-field detection path, effectively degrading the signal-to-background ratio. Several optimizations of LSFM have been implemented to mitigate or to avoid this effect. In particular, structured illumination (DSLM-SI)⁹ and confocal-like detection (CLSFM)¹⁰ have been introduced into the DLSM configuration to spatially filter the out-of-focus and scattered light. DSLM-SI requires multiple images and postprocessing, while CLSFM needs only to record line-by-line the fluorescence emitted at each light beam position through a confocal slit placed in the detection path. CLSFM can be implemented either by acquiring an image for each illumination position and then fusing them,⁷ or in a single acquisition by descanning with two additional galvanometric mirrors placed before and after a physical slit,¹¹ or by leveraging as a virtual slit the in-built rolling shutter readout mode of scientific complementary metal–oxide–semiconductor (sCMOS) cameras¹⁰ [Fig. 1(b)]. In the two latter cases, synchronizing the rates and positions of the digital light-sheet and the rolling shutter line-by-line sensor readout is enough to form the confocal image, which is simple and cost-efficient to realize, to the point of stimulating even camera manufacturers to implement a fine control of the rolling shutter parameters in their sensor drivers.

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.