SUMMARY Many visual animals have innate preferences for particular wavelengths of light, which can be modified by learning. Drosophila’s preference for UV over visible light requires UV-sensing R7 photoreceptors and specific wide-field amacrine neurons, called Dm8. Here we identify three types of medulla projection neurons downstream of R7 and Dm8, and show that selectively inactivating one of them (Tm5c) abolishes UV preference. Using a modified GRASP method to probe synaptic connections at the single-cell level, we reveal that each Dm8 neuron forms multiple synaptic contacts with Tm5c in the center of Dm8’s dendritic field, but sparse connections in the periphery. By single-cell transcript profiling and RNAi-mediated knockdown, we determine that Tm5c uses the kainate receptor Clumsy to receive excitatory glutamate input from Dm8. We conclude that R7s->Dm8->Tm5c form a hard-wired glutamatergic circuit that mediates UV preference by pooling ~16 R7 signals for transfer to the lobula, a higher visual center.

Summary Background In the fly’s visual motion pathways, two cell types - T4 and T5 - are the first known relay neurons to signal small-field direction-selective motion responses [1]. These cells then feed into large tangential cells that signal wide-field motion. Recent studies identified two types of columnar neurons in the second neuropil, or medulla, that relay input to T4 from L1, the ON-channel neuron in the first neuropil, or lamina, thus providing a candidate substrate for the elementary motion detector (EMD) [2]. Interneurons relaying the OFF-channel from L1’s partner, L2, to T5 are so far not known however. Results Here we report that multiple types of transmedulla (Tm) neurons provide unexpectedly complex inputs to T5 at their terminals in the third neuropil, or lobula. From the L2 pathway, single-column input comes from Tm1 and Tm2 and multiple-column input from Tm4 cells. Additional input to T5 comes from Tm9, the medulla target of a third lamina interneuron L3, providing a candidate substrate for L3’s combinatorial action with L2 [3]. Most numerous, Tm2 and Tm9’s input synapses are spatially segregated on T5’s dendritic arbor, providing candidate anatomical substrates for the two arms of a T5 EMD circuit; Tm1 and Tm2 provide a second. Transcript profiling indicates that T5 expresses both nicotinic and muscarinic cholinoceptors, qualifying T5 to receive cholinergic inputs from Tm9 and Tm2, which both express ChAT. Conclusions We hypothesize that T5 computes small-field motion signals by integrating multiple cholinergic Tm inputs using nicotinic and muscarinic cholinoceptors.