(A) Agarose-embedded 8 dpf zebrafish larvae were placed on an elevated stage, within a cylindrical recording chamber, and visually stimulated using a pico-projector. Spontaneous and visually induced neuronal activities were monitored by two-photon calcium imaging.

(B) An optical section of a larva’s brain pan-neuronally expressing GCaMP3 (HuC:GCaMP3). The optic tectum is highlighted in red. The rostro-caudal and medio-lateral axes of the right tectum are schematically outlined by dashed and pointed lines, respectively. Cb, cerebellum; H, habenula; Hb, hindbrain; OT, optic tectum; Te, telencephalon.

(C) Typical single-trial neuronal ΔF/F traces induced by 1 s visual stimulation with 4° light spots. Vertical gray line: stimulus onset. Stimulus azimuthal positions are indicated at each stimulation onset (0°, negative and positive angle values represent frontal, right, and left visual field positions, respectively). Significant ΔF/F transients are highlighted in red. Breaks in the traces are discarded frames due to movement artifacts.

(D) Representative neuronal spatial tuning curves (red line). Mean ± SD of neuronal responses are shown in black.

(E) Topography examples of neuronal groups consistently activated by light spots at the azimuth angles indicated in the top right corner. Activated neurons are colored in yellow, and the neuronal groups’ centroids are depicted as red asterisks. Bottom right: the neuronal group centroids color coded according to the azimuth angle (color bar on the right). As expected from a retinotopic map, a continuous progression of centroids along the contra-lateral rostro-caudal axis is clearly observed. Top left: anatomical landmarks. Dashed lines delineate the tectal-cerebellar and inter-hemispheric tectal boundaries. Cb, cerebellum; Np, tectal neuropil; SPV, tectal somatic periventricular layer.

(F) Raster plot of ΔF/F responses presented in (E). Grayscale, ΔF/F amplitude. Top labels indicate the light-spot azimuth angle. For visualization purposes, we show trials rearranged according to azimuth angle (during experiments, they were presented in random order). Only responsive neurons are shown. Bottom: histogram of the neuronal Ca2+ transients. Red bars mark light-spot stimulation periods.

(G) Normalized topography of neuronal groups induced by 4° light-spot stimulation experiments (n = 9). Each point in the upper panel represents the normalized position of a visually activated neuron, whose color indicates the absolute azimuth angle of the stimulating light spot (color bar on top). Bottom: probability density distributions of the normalized rostro-caudal neuron positions according to the stimulus position. Distributions are color coded according to the top color bar. Dots represent the medians. The smooth progression of the latter reflects a robust tectal retinotopic map conserved across larvae.