Nacre

Wildtype WIK larvae were used for behavioral experiments; for reticulospinal imaging, nacre fish on a WIK background were used; for two-photon imaging, nacre fish expressing GCaMP2 under control of the elavl3^{30 (link),20 (link)} promoter were used (previously known as HuC), again on a WIK background. All experiments were approved by Harvard University’s Standing Committee on the Use of Animals in Research and Training. Zebrafish larvae ages 6 to 7 dpf were anesthetized with MS222 and paralyzed by injection with a 1mg/ml bungarotoxin solution (Sigma-Aldrich), then suspended from structural pipettes (Suppl. Fig. S3), or embedded in agarose after which the agarose around the tail was removed. Motor nerve recordings were made with a Multiclamp 700B amplifier, simultaneously with two-photon imaging. Experiments were done at room temperature in filtered facility fish water. Visual scenes were projected onto a diffusive screen underneath the petri dish containing the fish via a mini projector, whose light source was replaced by a red Luxeon Rebel LED that was pulsed in synchrony with the fast scan mirror, so that illumination only occurred at the edges of the image where the scan mirror changed direction (typically at 800 Hz) to avoid any corruption of the two-photon images. Visual scenes consisted of square gratings with spatial period 12mm moving at 1cm/s from tail to head in the absence of motor nerve signals (−1 cm/s). When the processed swim signal was above an automatically set threshold (see Suppl. Meth. 1.3 and Suppl. Fig. S2), the locomotor drive was defined as the area underneath the curve of the processed swim signal during the current and previous video frame. The processed swim signal was defined to be the standard deviation of the raw swim signal in a sliding window of 15ms (see Fig. 1d). In the presence of such motor nerve signals, the instantaneous virtual fish velocity was set, 60 times per second (at the rate of the 60Hz projector), to −1cm/s + [gain] × [instantaneous locomotor drive], where the gain was set experimentally, after which the velocity decayed back to −1cm/s at a rate of 15cm/s², approximately matched to freely swimming fish dynamics (Supplementary figure S1). The high gain was chosen to be two to five times higher than the low gain and these values bracketed the ‘natural’ gain setting that described the transformation of motor activity into optic flow in a freely swimming fish. The high- and low-gain settings were manually adjusted for each fish, as different fish showed different ranges of adaptability. Some fish exhibited a transient increase in fictive motor output followed by a decrease after a gain-down change; these fish were discarded from the gain-down dataset because transient neural activity could not be distinguished from motor-related activity (rejection criterion: p < 0.03, paired t-test on fictive signal averages over seconds 0-15 versus averages over seconds 15-30 after gain-down change).

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.