5 and 6dpf AB/nacre larval zebrafish expressing GCaMP2, GCaMP3 or GCaMP5G under the elavl3 promoter were paralyzed by immersing them in 1 mg/ml solution of bungarotoxin dissolved in E3 fish embryo water and were subsequently embedded in 2% low melting point agarose in a 35 mm Petri dish. They were placed in a custom 2-photon microscope and imaged using a Mai Tai HP Ti-Sapphire laser tuned to 950 nm. The visual stimulus used for the experiment consisted of a light dot (0.5 mm × 0.5 mm) projected, using an amber (590 nm) LED mounted into a miniature LCOS projector, onto an opal glass screen directly underneath the larvae. Stimulus light was filtered with a narrow bandpass filter, and each fish was run through one stimulus set with the laser off to detect stimulus bleed-through, which was always negligible. The dot appeared to the left or right of the larva and moved on a straight line at a speed of 3 mm/s until it disappeared on the opposite side. The larva was located halfway along the dot’s trajectory and perpendicular to it, with the point of closest approach of the dot being 0.5 mm rostral to the larva.
The experimental protocol consisted of 1 min dark, followed by a presentation every 30 s of the moving dot, alternating between left to right and right to left. There were ten such presentations (five in each direction). The experiment concluded with 1 min dark, and therefore lasted 7 min in total. Individual frames were captured at 138.32 ms per frame (7.23 Hz), using a quad-interlaced scan pattern that ensured that each cell was sampled evenly at 4 times this frame rate.
Data analysis: Movies were assessed for x-y drift during the experiment (usually < 1 pixel), and a sub-pixel translation correction was applied using MATLAB software (David Heeger, NYU). Neuronal somata were detected based on their dark nuclei. Mean images were smoothed with a Gaussian, and local minima were detected. These were classified as cell nuclei if the ratio of the brightness 3 pixels from the center was more than 3.5 the brightness 1 pixel from the center, i.e. they look like a bright ring around a dark centre, and they were sufficiently bright (>17,500 photons detected per experiment). Fluorescence was then averaged over a 7×7 pixel square. Baseline fluorescence (F) was defined as the average fluorescence in the 50 frames immediately preceding each left-right stimulus.
The experimental protocol consisted of 1 min dark, followed by a presentation every 30 s of the moving dot, alternating between left to right and right to left. There were ten such presentations (five in each direction). The experiment concluded with 1 min dark, and therefore lasted 7 min in total. Individual frames were captured at 138.32 ms per frame (7.23 Hz), using a quad-interlaced scan pattern that ensured that each cell was sampled evenly at 4 times this frame rate.
Data analysis: Movies were assessed for x-y drift during the experiment (usually < 1 pixel), and a sub-pixel translation correction was applied using MATLAB software (David Heeger, NYU). Neuronal somata were detected based on their dark nuclei. Mean images were smoothed with a Gaussian, and local minima were detected. These were classified as cell nuclei if the ratio of the brightness 3 pixels from the center was more than 3.5 the brightness 1 pixel from the center, i.e. they look like a bright ring around a dark centre, and they were sufficiently bright (>17,500 photons detected per experiment). Fluorescence was then averaged over a 7×7 pixel square. Baseline fluorescence (F) was defined as the average fluorescence in the 50 frames immediately preceding each left-right stimulus.
