Defining the Connectivity and Dynamics of Peripheral Sour Taste Circuits

摘要 味觉是一个复杂的、综合的化学感觉系统，它引导着消费行为。味道 味蕾中的受体细胞（TRCs）不断更新，必须与周围味觉细胞重新连接， 神经元（GN）将味觉信号传递给大脑。外周味觉突触的更新和重建 对维持这个复杂的感觉系统至关重要研究表明， 控制TRCs的分化、成熟和周转，以及鉴定分子和 膝状体神经节神经元的功能分离群体。然而，突触连接和 由于缺乏研究技术，储税券和政府债券之间的动态仍然未知。到 为了克服这一点，我开发了跨突触伙伴的GFP重建（GRASP）来可视化突触 接触味蕾。GRASP由GRASP前和后分裂的GFP分子组成，其表达，定向，和 分别锚定在突触前和突触后细胞的突触膜中。因为， GRASP的组成部分可以通过遗传控制，我们可以用它来评估特定种群的连通性。 此外，GRASP在天然组织中产生明亮的荧光信号，这赠款了独特的研究能力。 在延时研究中的突触动力学。在初步工作中，我已经创造了转基因小鼠， 在PKD 2L 1启动子下的前GRASP蛋白的青色变体，其是酸性TRC的标志物。另外，我有 工程化的Flex-switch post-GRASP AAV，其使我们能够在GN中表达突触后GRASP。与 这项新技术，我的目标是从基因上识别潜在的GN-TRC突触伙伴， 特征突触动态味觉细胞周转期间，使用酸味TRCs作为模型。为了实现这些目标， 该提议将研究两个目标，1）鉴定真菌状中酸性TRC的突触伴侣， 和喉味蕾，以及2）确定酸性TRC突触的寿命和动力学， 时间本研究旨在利用GRASP这一新的创新技术来阐明神经元的突触功能， 周边味觉系统的连通性和动态性，这将在以下领域产生深远的影响： 味觉发育、连接和味觉障碍。

Abstract The sense of taste is a complex and integrative chemosensory system that guides consummatory behavior. Taste receptor cells (TRCs) in the taste bud are continuously renewed and must reconnect with peripheral gustatory neurons (GNs) to relay taste signals to the brain. The turnover and re-establishment of peripheral taste synapses is vital to maintain this complex sensory system. Studies have demonstrated the molecular mechanisms governing the differentiation, maturation and turnover of TRCs as well as the identification of molecularly and functionally separate populations of geniculate ganglion neurons. However, the synaptic connectivity and dynamics between TRCs and GNs remains unknown due to the lack of technologies to examine them. To overcome this, I have developed GFP Reconstitution Across Synaptic Partners (GRASP) to visualize synaptic contacts in the taste bud. GRASP consists of pre- and post-GRASP split GFP molecules expressed, directed, and anchored into the synaptic membranes of pre- and post-synaptic cells, respectively. Because expression of the GRASP components can be controlled genetically, we can use it to assess the connectivity of specific populations. Moreover, GRASP produces bright fluorescent signals in native tissues which grants the unique ability to study synapse dynamics in time-lapse studies. In preliminary work, I have created transgenic mice that express the cyan variant of the pre-GRASP protein under the PKD2L1 promoter, a marker for sour TRCs. Additionally, I have engineered Flex-switch post-GRASP AAVs which enables us to express the post-synaptic GRASP in GNs. With this novel technique at hand, my goal is to genetically identify potential GN-TRC synaptic partners and to characterize synaptic dynamics during taste cell turnover, using sour TRCs as a model. To achieve these goals, this proposal will investigate two aims, 1) Identify synaptic partners for sour TRCs in the fungiform, circumvallate, and laryngeal taste buds, and 2) Determine the longevity and dynamics of sour TRC synapses over time. This proposal is designed to use GRASP, a novel and innovative technique, to elucidate synaptic connectivity and dynamics in the peripheral taste system, which will have far-reaching impact in the fields of taste development, connectivity, and taste disorders.