Fast whole-brain imaging of seizures in zebrafish larvae by two-photon light-sheet microscopy

Abstract

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.

Quantity as a Fish Views It: Behavior and Neurobiology

“…The parallelized 2D image collection enables a longer signal integration time for each voxel, thus requiring a much-reduced peak laser intensity compared with 2p-PSM. Recent implementations of 2p-SPIM, as applied to brain-wide functional imaging of zebrafish (Wolf et al, 2015(Wolf et al, , 2017Keomanee-Dizon et al, 2020;de Vito et al, 2022), have achieved imaging frame rates of 20-150 Hz and volumetric rates spanning ∼1-5 Hz. Importantly, in these studies, the upper limit of the imaging rate is not dictated by the imaging hardware but rather by the putative threshold of photodamage, which starts to appear as the laser power is increased.…”

“…Here, again, the visual inertness of the 2-photon excitation light was critical to enable brain-wide imaging while the animals were treated to cycles of light/dark stimuli. Finally, 2p-SPIM was used by de Vito et al (2022) to map the whole-brain activity patterns of 4-dpf zebrafish undergoing epileptic seizures. The authors characterized the spatial-temporal dynamics of the pathological seizure activity and identified a previously-unknown caudo-rostral ictal wave pattern.…”

Power‐effective scanning with AODs for 3D optogenetic applications

“…Nonetheless, such a system can represent an add-on module for existing microscopes. Indeed, applying this AOD-based photostimulation head as an add-on to a whole-brain imaging system, such as a light-sheet fluorescence microscope [62,63], would open the way to numerous further experimental possibilities, toward all optical methods [64,65]. Fast axial-light addressing, large FOV and deep optical access, also guaranteed by 2P excitation, will help in opening new neuroscience scenarios.…”

Enhanced neuroimaging with a calcium sensor in the live adultDrosophila Melanogasterbrain using closed-loop adaptive optics light-sheet microscopy

“…The intrinsic optical sectioning of LSFM enables to image with high Signal-to-Background Ratio (SBR), over a large Field-of-View (FOV) using modern objectives, which also preserves a high spatial resolution at the single cell level. Starting from initial setups, various types of LSFM approaches have been reported [7, 8, 9], including improvements at the excitation path to provide a thin beam over a large FOV [10, 11, 12], and advanced setups mostly based on remote focusing enabling fast 3D imaging [13, 14, 15]. These new setups make LSFM undeniably one of the most recommended optical sectioning methods for high-speed imaging of 3D samples.…”