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. Using a combination of genetic, histological, electrophysiological and behavioral approaches, we study how the visual system processes chromatic information to guide behaviors in Drosophila. Using color preference and color learning assays, we demonstrated that flies innately prefer short wavelengths of light but they can be trained to select specific wavelengths of light by Pavlovian conditioning, indicating that flies, like honeybee and human, have true color vision. Using both light and electron microscope, we mapped the synaptic circuits of the chromatic photoreceptors, R7s and R8s, and their synaptic target neurons in the medulla ganglion of the peripheral visual system. We focused on the amacrine neurons, which interconnect medulla columns and the medulla projection (Tm) neurons, which are thought to serve functions analogous to those of vertebrate retinal ganglion cells by processing and relaying photoreceptor information to higher visual center. 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 Tm5a/b receive direct synaptic inputs from R7s while Tm9, Tm20 and Tm5c receive direct synaptic inputs from R8s. In addition, these Tm neurons receive indirect inputs from R1-6 via L3. These Tm neurons relay spectral information from the medulla to the higher visual center, the lobula. In addition to the direct pathways from photoreceptors to Tm neurons, the amacrine neuron Dm8 receives input from multiple R7s and provides input for Tm5a/b/c. To relate neural connectivity to functions, we assigned components of synaptic machinery to specific connections. To directly probe the usage of neurotransmitters and receptors which determine the polarity and dynamic of signal transmission, we developed a method to profile transcripts in single neurons. We used highly specific promoter-Gal4 constructs to label single types of neurons with GFP and to isolated these GFP-labeled neurons from adult fly brains and profiled their gene expression patterns by RT-PCR. Using this method, we determined that a large percentage of the first/second-order interneurons in the chromatic circuits express the vesicular glutamate transporter indicating that they are glutamatergic while the remaining chromatic circuits express choline acetyltransferase and therefore are likely cholinergic. This contrasts with the motion detection pathway, which is mostly cholinergic. By inactivating specific neurons and examining behavioral consequences, we 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. RNAi-knock-down of vesicular glutamate transporter in the Dm8 neurons significantly reduced UV preference, suggesting that glutamatergic output of Dm8 is critical for its functions. While Dm8 provides inputs for three types of transmedulla neurons, Tm5a/b/c, inactivating Tm5c alone abolished UV preference, indicating that Tm5c is the key downstream targets for this behavior. Furthermore, RNAi-knockdown of Kainate-type iGluR in Tm5c, thus inhibiting its ability to receive glutamatergic Dm8 inputs, significantly reduced UV preference. Using a modified GRASP (GFP-reconstitution across synaptic partners) method, which allows single-cell analysis of bona fide synaptic connections, we demonstrated that Dm8 receives 16 R7 inputs and provides inputs for 1-2 Tm5c neurons in the center of Dm8's receptive field. Thus, the R7s->Dm8->Tm5c connections constitute a hard-wired pooling circuit for detecting dim UV light. To map the circuits that transform photoreceptor signals into color percepts, we developed a novel aversive operant conditioning assay for intensity-independent color discrimination. Single flying flies were magnetically tethered in an arena surrounded by blue/green LEDs and the flies are then conditioned to discriminate between equiluminant blue or green stimuli. Wild-type flies can be trained in this paradigm 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. Genetically inactivating four classes of first-order interneurons, Tm5a/b/c and Tm20, abolishes color learning. However, inactivating subsets of these neurons is insufficient to block color learning, suggesting that true color vision is mediated by multiple redundant pathways. The apparent redundancy in learned color discrimination sharply contrasts with innate spectral preference, which is dominated by a single pathway. Using a similar strategy, we mapped the visual motion pathways in Drosophila. Previous studies revealed that two types of lobula plate neurons, T4 and T5, signal small-field direction-selective motion responses and are downstream of the first-order neurons, L1 and L2, or the ON- and OFF-channel neurons, respectively. Serial-EM reconstruction revealed that T5 receives direct synaptic inputs from four types of transmedulla neurons, Tm1, Tm2, Tm4, and Tm9. Our transcript profiling and immunohistochemistry revealed that these Tm neurons express choline acetyltransferase and therefore are likely cholinergic. Furthermore, we found that T5 expresses both nicotinic and muscarinic acetylcholine receptors. The Tm2 and Tm9 input synapses are spatially segregated on T5s dendritic arbor, thus providing candidate anatomical substrates for the two arms of elementary motion detector circuits. Based on the synaptic circuit and receptor expression profiles, we hypothesize that T5 computes small-field motion signals by integrating multiple cholinergic Tm inputs using nicotinic and muscarinic cholinoceptors.
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