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

神经回路允许有机体感知环境中的刺激并产生适当的行为 回应。在味觉上，这些行为是在评估食物的营养含量时被唤起的 消息来源。甜的和苦的食物分别在昆虫和昆虫身上引起吸引和厌恶的反应。 哺乳动物。然而，人们对味觉回路和神经机制知之甚少。 在感官细胞之外辨别甜味和苦味。在老鼠身上进行研究的证据表明 这种味道的品质，如甜味和苦味，是通过标签线模型进行处理的。在这个模型中，甜蜜和 苦味者是由平行和分离的电路代表的。监测全脑神经元的研究 果蝇在甜味和苦味刺激下的活动表明，标记线模型也是 在飞行中行动。然而，在逐层的基础上对味觉电路进行系统评估是 需要充分评估果蝇甜味和苦味品质的编码机制。 我们实验室开发了一种新的神经回路标测和操作方法--TRANS-Tango 在果蝇身上。利用反式探戈，我们已经确定了突触后对甜味和苦味的味觉投射 感觉细胞--回路中的二级神经元。这一分析揭示了广泛的解剖学 两个赛道之间的相似之处，但需要更精细的比较来评估甜蜜的程度 苦涩的赛道汇聚在一起。这项提案详细说明了解决甜蜜和痛苦的三管齐下的方法 电路映射到单细胞水平，并根据味觉刺激的功能反应对味觉投射进行分类 和细胞类型。为了实现这一点，我开发了几种新的跨探戈介导的神经元策略 侧写。首先，我将描述二级神经元的形态，并制作一份味觉图谱 通过随机标记和配准到模板大脑，具有单细胞分辨率的投影。第二，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 融合到二级神经元中的核定位序列，以监测它们对味觉的活动 刺激。最后，我将概述甜味和苦味回路中二阶神经元的细胞类型 绿色荧光蛋白与核膜蛋白的二级融合表达 神经元的细胞核提纯和转录分析。拟议的实验将导致 甜味和苦味回路中二阶神经元的全面重建，并最终 阐明果蝇大脑用来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病媒生物，更好地了解它们的味觉回路将 对全球人类健康有重大影响。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。

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.