Two-photon calcium recordings of cones

For colour vision, retinal circuits separate information about intensity and wavelength. This requires circuit-level comparison of at least two spectrally distinct photoreceptors. However, many vertebrates use all four ‘ancestral’ photoreceptors (‘red’, ‘green’, ‘blue’, ‘UV’), and in those cases the nature and implementation of this computation remains poorly understood. Here, we establish the complete circuit architecture of outer retinal circuits underlying colour processing in the tetrachromatic larval zebrafish, which involves all four ancestral cone types and three types of horizontal cells. Our findings reveal that the synaptic outputs of red- and green-cones efficiently rotate the encoding of natural daylight in a principal component analysis (PCA)-like manner to yield primary achromatic and spectrally-opponent axes, respectively. Together, these two cones capture 91.3% of the spectral variance in natural light. Next, blue-cones are tuned so as to capture most remaining variance when opposed to green-cones. Finally, UV-cones present a UV-achromatic axis for prey capture. We note that fruit flies – the only other tetrachromat species where comparable circuit-level information is available - use essentially the same strategy to extract spectral information from their relatively blue-shifted terrestrial visual world. Together, our results suggest that rotating colour space into primary achromatic and chromatic axes at the eye’s first synapse may be a fundamental principle of colour vision when using more than two spectrally well-separated photoreceptor types.

Methods
The data was collected from zebrafish larvae (7 days post-fertilisation) expressing SyGCaMP6f in each cone type. SyGCaMP6f fluorescent images during visual stimulation were recorded on a custom built two-photon microscope. Detailed information is in the manuscript.