Characterizing the Drosophila taste circuits with next-generation trans-Tango strategies

用下一代跨探戈策略表征果蝇味觉回路

基本信息

批准号：
10391324
负责人：
Anthony Michael Crown
金额：
$ 4.68万
依托单位：
BROWN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-03-01 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10391324
关键词：
ARNT gene Address Affect Agriculture Anatomy Animals Atlases Aversive Stimulus Behavior Behavioral Brain Calcium Cell Nucleus Cells Code Communicable Diseases Confocal Microscopy Dengue Dental crowns Detection Disease Vectors Drosophila genus Drosophila melanogaster Environment Esthesia Evaluation Food Future Gene Expression Profile Genetic Green Fluorescent Proteins Health Heterogeneity Human Individual Insect Vectors Insecta Label Laboratories Ligands Logic Malaria Mammals Maps Mediating Membrane Proteins Mentors Methods Modality Modeling Molecular Monitor Morphology Mus Nervous system structure Neural Pathways Neurons Neurosciences Research Nuclear Nuclear Envelope Nuclear Inner Membrane Nuclear Localization Signal Nutritional Organism Output Pathway interactions Pattern Poison Population Process Proteolysis RNA Receptor Activation Receptor Cell Reporter Research Resolution Sensory Signal Pathway Signal Transduction Site Source Stimulus Synapses Taste Perception Techniques Testing Tissue-Specific Gene Expression Training Variant Visualization West Nile virus Work behavioral response calcium indicator career cell type experimental study fly food shortage information processing neural circuit neuromechanism next generation next generation sequencing novel programs receptor reconstruction relating to nervous system response sensor skills taste stimuli technological innovation tool transcription factor transcriptome transcriptomics two photon microscopy

项目摘要

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.

神经回路使生物体可以在其环境中感知刺激并产生适当的行为回答。从品味的意义上讲，这些行为在评估食物的营养含量时会引起人们的兴趣来源。甜食和苦涩的食物分别在昆虫和哺乳动物。但是，对于味觉感觉和神经机制的电路知之甚少区分感觉细胞以外的甜和苦味味。小鼠研究的证据暗示这种味道质量（例如甜和苦涩）是通过标记的线模型来处理的。在这个模型中，甜蜜苦味剂以平行和隔离的电路表示。研究监测脑范围的神经元果蝇中用甜和苦味剂刺激时的活动表明，标记的线模型也是飞行中的手术者。但是，按一层对味觉电路进行系统评估是需要充分评估果蝇中甜和苦味品味的编码机制。我们的实验室开发了Trans-Tango，这是一种用于神经电路映射和操纵的新方法在果蝇中。使用trans-tango，我们已经确定了味道后的味道预测感觉细胞 - 电路中的二阶神经元。该分析揭示了广泛的解剖学这两个电路之间的相似性，但是需要进行更精确的比较来评估甜度的程度和苦路汇聚。该提案详细介绍了一种解决甜蜜和苦涩的三管齐下的方法电路图达到单细胞水平，并通过对味觉刺激的功能响应来对味觉投影进行分类和细胞类型。为了实现这一目标，我已经开发了几种新型的反式tango介导的神经元策略分析。首先，我将描述二阶神经元的形态，并发展出味道的地图集通过随机标记和对模板大脑的注册进行单细胞分辨率的投影。第二，我将通过表达钙传感器来确定这些神经元对各种味觉刺激的响应性融合到二阶神经元中的核定位序列，以监测其味道的活性刺激。最后，我将通过甜蜜和苦的电路中的二级神经元的细胞类型来介绍在二阶中表达融合到核膜蛋白的绿色荧光蛋白（GFP）纯化其核和转录组分析的神经元。提出的实验将导致在甜和苦的电路中综合重建二阶神经元，最终阐明果蝇大脑使用的编码模型来处理口味信息。此外，这些研究将揭示果蝇中的味道电路是否遵循与哺乳动物相似的逻辑。因为昆虫是农业和常见疾病媒介的主要害虫，对它们的味觉更好的了解将对全球人类健康有重大影响。最后，该研究计划是培训计划的核心包括开发专业技能的活动，以准备安东尼·皇冠（Anthony Crown）为学术研究的职业生涯。