Defining the Connectivity and Dynamics of Peripheral Sour Taste Circuits

摘要 味觉是指导消费行为的复杂、综合的化学感觉系统。味觉 味蕾中的受体细胞不断更新，必须与周围的味觉重新连接 神经元(GN)将味觉信号传递给大脑。外周味觉突触的周转与重建 对维持这个复杂的感官系统至关重要。研究已经证明了分子机制。 调控TRCs的分化、成熟和周转，以及分子和 功能上分离的膝状神经节神经元群体。然而，突触连接和 由于缺乏审查技术，TRCS和GNS之间的动态仍然不得而知。至 克服了这一点，我开发了跨突触合作伙伴的GFP重构(GRASP)来可视化突触 在味蕾中接触。GRAPH由表达、定向和表达的前后裂解的GFP分子组成 分别锚定在突触前和突触后细胞的突触膜上。因为表达了 GRAPH组分可以从基因上控制，我们可以用它来评估特定人群的连接性。 此外，GRAP在自然组织中产生明亮的荧光信号，这赋予了研究的独特能力 时移研究中的突触动力学。在前期工作中，我已经创造了表达这种基因的转基因小鼠 PKD2L1启动子下的前抓取蛋白的青色变体，是酸性TRCs的标志。另外，我还有 工程化的Flex-Switch后抓握AAVs，使我们能够在GNS中表达突触后抓握。使用 这项手头的新技术，我的目标是从基因上识别潜在的GN-TRC突触伙伴并 以酸味细胞为模型，描述味觉细胞周转过程中突触的动态变化。为了实现这些目标， 这项提案将调查两个目标，1)为真菌样中的酸性TRCs寻找突触伙伴， 2)决定酸性TRC突触的寿命和动力学。 时间到了。这项提议旨在使用GRAPH这一新颖而创新的技术来阐明突触 外围味觉系统的连接性和动态性，这将在以下领域产生深远影响 味觉发育、连通性和味觉失调。

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.