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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.

神经回路允许生物体感知环境中的刺激并产生适当的行为反应。应答在味觉上，这些行为是在评估食物的营养成分时诱发的源头甜的和苦的食物分别在昆虫和昆虫中引起吸引和厌恶的反应。哺乳动物然而，人们对味觉的回路和味觉的神经机制知之甚少。区分甜味和苦味的味觉细胞。小鼠研究的证据表明，通过标记线模型处理诸如甜和苦的味道质量。在这个模型中，甜蜜和促苦味剂由平行和分离的回路表示。监测全脑神经元的研究果蝇中甜味剂和苦味剂刺激后的活性表明，标记的线模型也是在飞行中的操作。然而，在逐层基础上对味觉回路的系统评估是不可能的。需要充分评估果蝇中甜味和苦味品质的编码机制。我们的实验室开发了trans-Tango，一种用于神经回路映射和操作的新方法在果蝇中。使用trans-Tango，我们已经确定了甜和苦的突触后味觉投射感觉细胞-回路中的二级神经元。这项分析揭示了广泛的解剖学这两个电路之间的相似性，但需要更精细的比较来评估甜蜜的程度。和痛苦的回路汇聚在一起这份建议书详细介绍了三管齐下的办法，以解决甜与苦电路映射到单细胞水平，并根据对味觉刺激的功能反应对味觉投射进行分类细胞类型。为了实现这一目标，我开发了几种新的trans-Tango介导的神经元侧写首先，我将描述二级神经元的形态学特征，并开发一个味觉图谱通过随机标记和配准到模板大脑的具有单细胞分辨率的投影。二我将通过表达钙传感器来识别这些神经元对各种味觉刺激的反应性融合到二级神经元的核定位序列上，以监测它们在味觉上的活动刺激.最后，我将描述甜味和苦味回路中二级神经元的细胞类型，表达与核膜蛋白融合的绿色荧光蛋白（GFP），神经元用于纯化其细胞核和转录组学分析。拟议的实验将导致一个全面重建甜味和苦味回路中的二级神经元，阐明果蝇大脑处理味觉信息的编码模型。此外，这些研究将揭示果蝇的味觉回路是否遵循与哺乳动物相似的逻辑。因为昆虫是农业中的主要害虫和常见的疾病媒介，更好地了解它们的味觉回路将对全球人类健康产生重大影响。最后，这项研究计划是培训计划的核心，包括培养专业技能的活动，为Anthony Crown从事学术研究做好准备。