Most visual animals utilize spectral information in two major ways. Color vision, which differentiates spectral compositions independent of brightness, provides animals, from insects to primates, great power for object recognition and memory registration and retrieval. Innate spectral preference, on the other hand, is intensity-dependent, and reflects individual species' specific ecological needs. Using a combination of genetic, histological, electrophysiological and behavioral approaches, we study how visual circuits process chromatic information to guide behaviors in Drosophila. The receptor mechanism for color vision and spectral preference is well understood but post-receptor processing of chromatic information is poorly understood. Our strategy is (i) to identify key neuronal types and to map their synaptic connections, (ii) to examine functional requirement of identified neurons for color vision and spectral preference behaviors, and (iii) to determine the neurotransmitter and receptor systems utilized in synaptic circuits. By molecular genetics and histology, we mapped the synaptic circuits of the chromatic photoreceptors, R7s and R8s, and their synaptic target neurons, Tm and Dm neurons, in the peripheral visual system. The medulla projection (Tm) neurons, analogous to vertebrate retinal ganglion cells, relay photoreceptor information to higher visual centers while the Dm neurons connect photoreceptors to Tm neurons. Using a modified GRASP (GFP reconstitution across synaptic partners) method, we characterized the synaptic connections between photoreceptors and the Tm/Dm neurons. We found that the chromatic photoreceptors, R7 (UV-sensing) and R8 (blue/green-sensing), provide inputs to a subset of first-order interneurons. The first-order interneurons Tm9, Tm20 and Tm5c receive direct synaptic inputs from R8s while Tm5a/b receive direct synaptic inputs from R7s. These Tm neurons relay spectral information from the medulla to the deep visual processing center, the lobula. In addition to the direct pathways from photoreceptors to Tm neurons, the amacrine neuron Dm8 receives inputs from multiple R7s and provides input for Tm5a/b/c. To assign neurons to innate spectral preference, we systematically inactivated different first-order interneurons and examined behavioral consequences. We have previously found that the amacrine Dm8 neurons, which receive UV-sensing R7 photoreceptor inputs, are both required and sufficient for animals' innate spectral preference to UV light. We now found that inactivating Tm5c, one of Dm8s synaptic targets, abolished UV preference, indicating that Tm5c is the key downstream targets for spectral preference. Using the single-cell transcript profiling technique, we found that both Dm8 and Tm5c express vesicular glutamate transporter (VGlut), suggesting that they are glutamatergic. RNAi-knockdown of VGlut in Dm8 or Tm5c significantly reduced UV preference, suggesting the critical functions of the glutamatergic output of Dm8 and Tm5c. We further identified that Tm5c expresses four kainite-type glutamate receptors and that RNAi-knockdown of these receptors, thus preventing Tm5c from receiving Dm8 inputs, significantly reduced UV preference. Thus, the R7s->Dm8->Tm5c connections constitute a hard-wired glutamatergic circuit for detecting dim UV light. To identify color-vision circuits, we developed a novel aversive operant conditioning assay for intensity-independent color discrimination. We conditioned single flies to discriminate between equiluminant blue or green stimuli. We found that wild-type flies can be trained to avoid either blue or green while mutant lacking functional R7 and R8 photoreceptors can not, indicating that the color entrainment requires the function of the narrow-spectrum photoreceptors R7s and/or R8s. Inactivating all four types of first-order interneurons, Tm5a/b/c and Tm20, abolishes color learning. However, inactivating different subsets of these neurons is insufficient to block color learning, suggesting that true color vision is mediated by parallel pathways with redundant functions. The apparent redundancy in learned color discrimination sharply contrasts with innate spectral preference, which is dominated by a single pathway, R7->Dm8->Tm5c. To determine how color information is processed in the higher visual center, we set out to identify the lobula neurons that receive direct synaptic inputs from the chromatic Tm neurons, Tm5a/b/c and Tm20. We first collected and characterized available Gal4 lines for lobula expression. Second, we used our modified GRASP method to examine potential contacts between chromatic Tm neurons and the dendrites of candidate lobula neurons. We identified four types of lobula neurons that form synaptic contacts with chromatic Tm neurons: two novel lobula intrinsic neurons, Li3 and Li4, and two lobula projection neurons, LT11 and LC14. Each LT11 elaborates a large dendritic tree to cover the entire lobula layers, Lo4-6, and projects an axon to optic glomeruli in the central brain. Each Li4 extends dendrites to cover about 60% of the lobula layer, Lo5. Using the single-cell GRASP method we developed, we further characterized the synaptic connections at the single-cell resolution and found that both Li4 and LT11 neurons receive inputs from all four chromatic Tm neurons. Our anatomical study suggests that the first relay in the lobula involves both spatial and chromatic integration.
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