Development of optical methods for real-time whole-brain functional imaging of zebrafish neuronal activity

In this PhD thesis, we performed functional imaging of zebrafish larvae with a multi-modal and multi-scale approach. In particular, we focused our research on measuring neuronal activity in both physiological and pathological conditions. We adopted a pharmacological model of epilepsy, by administering pentylentetrazole at different concentrations and inducing seizures of different entity in zebrafish larvae expressing in all CNS neurons the Ca2+ reporter GCaMP6s. Owing to the relation between neuronal activity (i.e. action potentials) and intracellular Ca2+ concentration, we were able to measure neuronal activity by recording the changes in fluorescence of GCaMP indicator. Using a custom-made widefield fluorescence microscope, we measured activity during the onset and propagation of seizures, investigating the dynamics between different brain regions along with tail locomotor activity. We implemented commonly used zebrafish high-throughput drug screening assays, measuring only behavioural parameters (i.e. velocity of swimming, total travelled length), with a direct measure of the overall brain activity, thus laying the foundation for novel drug screening methods capable of improved efficacy. In order to improve the spatio-temporal resolution of brain activity recordings, being able to perform optical sectioning of the transparent zebrafish brain, we performed Bessel beam illumination light-sheet fluorescence microscopy measurements. Indeed, with respect to conventional Gaussian illumination, Bessel beams, owing to their nondiffractive and self-healing properties, allow for a substantial reduction of haemodynamic artefacts jeopardizing functional recordings in conventional measurements. We applied a custom analysis pipeline to produce 3D maps of neuronal activity, with single cell resolution. Finally, in order to perform real-time whole-brain measurements with singleneuron resolution, we devised a novel two-photon light-sheet microscope able to image the entire larval brain at 1 Hz. Applying a pixel-wise custom analysis we were able to identify functional circuitries involved both in physiological and pathological neuronal communication.