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.

The intricate macroscopic and microscopic structure of the human brain poses a significant challenge for techniques that rely on diffuse near-infrared light for functional imaging and diagnostics.¹ The complex and heterogeneous composition of brain tissue influences light propagation, affecting the accuracy of these techniques. Current optical methods, such as functional near-infrared spectroscopy (fNIRS) and diffuse optical tomography (DOT), focus primarily on monitoring hemodynamic responses by mapping blood oxygenation changes in different brain regions.^{2 – 5} Among these, time-resolved techniques play a crucial role in extracting absolute tissue oxygen saturation values and estimating the reduced scattering coefficient,⁶ both of which are essential for functional and clinical assessments. A key advantage of these methods is their ability to separate extra-cerebral and cerebral contributions, as different photon propagation times correspond to different penetration depths. This feature enhances the accuracy of functional imaging and disease diagnostics by isolating signals from deeper brain structures, as long as light transport through brain tissue is accurately modeled.

Analyzing the structure of neuronal fibers with single axon resolution, in large volumes, remains an unresolved challenge in connectomics. Here, we propose MAGIC (Myelin Autofluorescence imaging by Glycerol Induced Contrast enhancement), a simple tissue preparation method to perform label-free fluorescence imaging of myelinated fibers. We demonstrate its broad applicability by performing mesoscopic reconstruction at sub-micron resolution of mouse, rat, monkey, and human brain samples and by quantifying the different fiber organization in Control and Reeler mouse’s hippocampal sections.