No abstract

Larval zebrafish ( Danio rerio ) are an ideal organism to study color vision, as their eye possesses four types of cone photoreceptors, covering most of the visible range and into the UV [1,2] . Additionally, their entire eye and nervous system are accessible to imaging, given they are naturally transparent [3][4][5] . Relying on this advantage, recent research has found that, through a set of color specific horizontal, bipolar and retinal ganglion cells (RGCs) [6][7][8] , the eye then relays tetrachromatic information to several retino-recipient areas (RAs) [9,10] . The main RA is the optic tectum, receiving 97% of the RGC axons via the neuropil mass termed Arborization Field 10 (AF10) [11,12] . In this work, we aim to understand the processing of color signals at the interface between RGCs and their targets in the brain. We used 2-photon calcium imaging to separately measure the responses of RGCs and neurons in the dorsal brain to stimulation with four different colors in awake animals. We find that color information is widespread throughout the larval brain, with a large variety of color responses among RGCs, and an even greater diversity in their targets. Specific combinations of response types are localized to specific nuclei, but we observe no single color processing structure. In the main interface in this pathway, the connection between Arborization Field 10 and the tectum, we observe key elements of color processing such as enhanced signal decorrelation and improved decoding [13,14] . Finally, when presenting a richer set of stimuli, we identify parallel processing of color, motion and luminance information in the same cells/terminals, evidence of a rich color vision machinery in this small vertebrate brain. Stimulation with different colors evokes diverse responses in RGCs and central brainOur goal is to characterize the flow of color information in the larval zebrafish brain, from the incoming RGCs and into the areas receiving these connections in the central brain. To that end, we used a custom-built microscope and 4-channel projector, capable of arbitrary color presentation ( Fig. 1A-B, Supp. Fig. 1A) to deliver full-field, sinusoidal intensity modulated stimuli 1 of four colors (Fig. 1C). The light sources were selected to broadly sample the range of wavelengths the fish is sensitive to at naturalistic intensities [6] , rather than attempting to isolate and stimulate individual cone types [15] . Projection was from the bottom to stimulate the dorsal retina, which is well suited for color computations as it concentrates many color opponent RGC responses [8] . We utilized two transgenic fish lines, one expressing the fluorescent calcium indicator GCaMP6s [16] in RGC axon terminals, and the other expressing the same indicator in most of the larval central brain (Fig. 1D). Figure 1. Stimulation with different colors evokes a variety of responses in zebrafish RGCs and RAs. A: schematic of the 2-photon microscope used for stimulation, projecting color stimuli from the bottom using a custom-designed DLP pr...