To understand how color information is processed in the fly brain, we set out (i) to identify the neurons constituting the chromatic circuits and their synaptic connections and, (ii) to determine their functions using various behavioral paradigms. To achieve the first objective, we first identified the first-order interneurons in the medulla ganglion, which receive direct synaptic inputs from the chromatic channels, R7 and R8. We exploited the knowledge that the photoreceptor neurons are histaminergic and therefore their synaptic target must express histamine receptors, which are histamine-gate chloride channels (encoded by the ort and HisCl1 genes). Using an ort-Gal4 driver, we identified the frist-order interneurons, which include two projection neurons, Tm5 and Tm9, as well as an amacrine neuron Dm8. Using serial EM reconstruction, we determined the synaptic circuits of these neurons. We found that (i) the amacrine neuron Dm8 receive inputs from multiple R7 and relay the information to the projection neurons, and (ii) the projection neurons, Tm5 and Tm9, receive direct synaptic inputs from the chromatic channels, UV-sensing R7s and Blue/Green-sensing R8s, respectively, as well as hitherto unsuspected indirect input from the achromatic channel via L3 (R1-R6). Thus, the projection neurons integrate both chromatic and achromatic channels and relay the information to the higher visual center, the lobula. The integration of multiple channels at the level of first- or second-order interneurons in flies is similar to that observed in the primate system of color-opponency, suggesting a convergent color-coding solution to a common problem in both visual systems.? To determine the function of these first-order interneurons for the second objective, we used a behavioral assay, called spectral preference test. In this behavioral paradigm, flies are tested for their innate preference for UV light over green light and this behavior has been known to depend on the UV-sensing R7s. We found that mutant flies lacking Ort, but not those lacking the other histamine receptor HisCl1, exhibited aberrant preference for green light. The ort phenotype could be phenocopied by inactivating Ort-expressing neurons, indicating that Ort-expressing neurons are required for UV preference. ? To determine the specific neurons that mediate this behavior, we subdivided the Ort-expressing neurons into several categories based on their use of neurotransmitters. Using a combinatorial gene expression system called the split-Gal4 system, we found that the projection neurons Tm5 and the amacrine neurons Dm8 are glutaminergic while the projection neurons Tm9 are cholinergic. By restoring Ort expression in specific neuron subsets, we found that the glutaminergic Tm5 and Dm8 neurons are sufficient to drive UV preference while the cholinergic Ort-expression neurons, including Tm9, are sufficient to drive green preference. In addition, inactivating glutaminergic Ort-expressing neurons abolished UV preference, indicating that they are required for UV preference. These results suggest that Tm5 and Dm8 relaying the information from the UV-sensing R7s to the lobula while Tm9 relaying blue/green-sensing R8s to the lobula. We further refine the Gal4 expression pattern by dividing the ort promoter into three highly conserved regions, C1-C3. We found that the glutaminergic Ort-C2 subset, which includes primarily Dm8 neurons, is both required and sufficient for conferring a flys preference to UV. Similar to the vertebrate amacrine cells which link bipolar to retina ganglion cells, Drosophila Dm8 neurons receive main synaptic input from R7s and provide input to the projection neuron Tm5. The importance of these connections is underscored by the finding that these indirect connections, from R7, to Dm8, then to Tm5, are both necessary and sufficient to drive UV preferences. We propose that Dm8 sacrifices spatial resolution for sensitivity by relaying signals from multiple R7s to small-field projections neurons. We note that Ort-expressing neurons do not include any Dm8-like wide-field neurons for R8s, and restoring activity in Tm9 projection neurons is sufficient to confer stronger green preference in ort mutants. Thus we speculate that Dm8 evolved uniquely to meet the ecological need to detect dim UV in the background of ample visible light.
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