Neural circuits allow an organism to sense stimuli in its environment and generate the appropriate behavioral responses. In the sense of taste, these behaviors are evoked upon assessing the nutritive content of a food source. Sweet and bitter foods elicit attractive and aversive responses, respectively, in both insects and mammals. However, little is known about the circuits for taste sensation and the neural mechanism for discriminating sweet and bitter tastants beyond the sensory cells. Evidence from studies in the mouse implies that taste quality, such as sweet and bitter, is processed through a labeled line model. In this model, sweet and bitter tastants are represented by parallel and segregated circuits. Studies monitoring brain-wide neuronal activity upon stimulation with sweet and bitter tastants in Drosophila suggest that a labeled line model is also operative in the fly. However, a systematic evaluation of the gustatory circuits on a layer by layer basis is required to fully evaluate the coding mechanism of sweet and bitter taste qualities in Drosophila. Our laboratory has developed trans-Tango, a new method for neural circuit mapping and manipulation in Drosophila. Using trans-Tango, we have identified the taste projections post-synaptic to the sweet and bitter sensory cells - the second-order neurons in the circuits. This analysis has revealed broad anatomical similarities between the two circuits, but a finer comparison is required to assess the degree to which the sweet and bitter circuits converge. This proposal details a three-pronged approach to resolve the sweet and bitter circuit maps to a single-cell level and classify the taste projections by their functional responses to taste stimuli and cell type. To achieve this, I have developed several novel trans-Tango-mediated strategies for neuronal profiling. First, I will characterize the morphology of the second-order neurons and develop an atlas of the taste projections with single-cell resolution through stochastic labeling and registration to a template brain. Second, I will identify the responsivity of these neurons to various classes of taste stimuli by expressing a calcium sensor fused to a nuclear localization sequence in the second-order neurons to monitor their activity upon taste stimulation. Finally, I will profile the cell types of the second-order neurons in the sweet and bitter circuits by expressing a green fluorescent protein (GFP) fused to a nuclear membrane protein in the second-order neurons for purification of their nuclei and transcriptomic analysis. The proposed experiments will result in a comprehensive reconstruction of the second-order neurons in the sweet and bitter circuits, and ultimately elucidate the coding model used by the Drosophila brain to process taste information. Further, these studies will reveal whether the gustatory circuitry in Drosophila follows a similar logic as in mammals. Because insects are major pests in agriculture and common disease vectors, better understanding of their gustatory circuits will have a major impact on global human health. Finally, this research program is at the core of a training plan that includes activities to develop professional skills for preparing Anthony Crown to a career in academic research.

Insects are a major human health concern since they act as vectors for diseases like West Nile virus, malaria, and dengue. Additionally, insect pests in agriculture cause massive losses of crop yield that contribute significantly to food shortages in the developing world. Therefore, understanding how insects enact attractive and aversive behaviors upon taste stimulation will have a major impact on global human health.