Electrophysiology of Taste Transduction

描述（由申请人提供）：当味觉刺激与受体细胞的顶膜相互作用时，味觉传导开始，引起细胞内Ca 2+增加。这最终导致递质的释放和味觉传入纤维的激活。已经鉴定了几种G蛋白偶联的味觉受体：T2 R对苦味有反应，T1 R2/T1 R3对甜味有反应，T1 R1/T1 R3和味觉mGluR 4对鲜味刺激有反应。此外，这些受体都激活PLC β 2，导致Ca 2+从细胞内储存释放和Ca 2+通过阳离子通道TrpM 5流入，尽管这些信号传导组分中的每一种的确切作用尚不清楚。令人惊讶的是，PLC β 2主要存在于II型味觉细胞中，其缺乏与传入神经纤维的常规突触。因此，这些味觉品质如何传递到神经系统尚不清楚。我们的目标是拟议的实验是阐明之间的联系的初始事件的味觉转导和传入神经纤维的激活。实验提出确定T1 R1/T1 R3和taste-mGluR 4在鲜味信号传导中的相对作用，它们的下游信号传导效应器，以及对鲜味和苦味刺激的反应如何传递给神经系统。目的1用免疫细胞化学和原位杂交技术确定两种鲜味受体是否共定位，用Ca 2+成像和膜片钳记录技术确定每种受体的反应模式，以及这种反应是否被5 '-核糖核苷酸增强，以及电导是否受到影响。目的2利用Ca 2+成像和电生理学来确定对鲜味和苦味转导重要的下游信号传导效应物，包括PLC β 2、TrpM 5和α-味蛋白的作用。在目标3中，我们将测试III型味觉细胞是否需要将苦味，鲜味或甜味信息传递到神经系统，并产生两个新的转基因小鼠。两者都涉及将基因靶向BDNF基因座，因为BDNF选择性地在具有突触的成年味觉细胞中表达：（1）BDNF-GFP小鼠，其将在所有具有突触的味觉细胞中表达GFP，因此它们可以被识别用于生理记录，以及（2）BDNF-DT小鼠，其将经历突触连接的味觉细胞的选择性消融，因此可以直接确定它们在味觉信息处理中的作用。

DESCRIPTION (provided by applicant): Taste transduction begins when sapid stimuli interact with the apical membrane of receptor cells, causing an increase in intracellular Ca 2+. This ultimately results in release of transmitter and activation of gustatory afferent fibers. Several G protein-coupled taste receptors have been identified: T2Rs respond to bitter, T1R2/T1R3 to sweet, and T1R1/T1R3 and taste-mGluR4 to umami stimuli. Further, these receptors all activate PLCbeta2, causing Ca2+ release from intracellular stores and Ca2+ influx via the cation channel TrpM5, although the precise role of each of these signaling components is not understood. Surprisingly, PLCbeta2 is present primarily in Type II taste cells, which lack conventional synapses with afferent nerve fibers. Thus, how these taste qualities are transmitted to the nervous system is unclear. Our goal for the proposed experiments is to elucidate the link between the initial events of taste transduction and activation of afferent nerve fibers. Experiments are proposed to determine the relative role of T1R1/T1R3 and taste-mGluR4 in umami signaling, their downstream signaling effectors, and how responses to umami and bitter stimuli are communicated to the nervous system. Aim 1 uses immunocytochemistry and in situ hybridization to determine whether the two umami receptors are co-localized, and Ca 2+ imaging and patch clamp recording to determine the response profile of each receptor, whether the response is potentiated by 5'-ribonucleotides, and the conductance(s) that are affected. Aim 2 utilizes Ca2+ imaging and electrophysiology to determine the downstream signaling effectors important for umami and bitter taste transduction, including the roles of PLCbeta2, TrpM5, and alpha-gustducin. In aim 3, we will test whether Type III taste cells are required for communicating bitter, umami, or sweet taste information to the nervous system with the production of two new transgenic mice. Both will involve targeting of genes to the BDNF locus, since BDNF is selectively expressed in adult taste cells with synapses: (1) BDNF-GFP mice, which will express GFP in all taste cells with synapses, so they can be identified for physiological recording, and (2) BDNF-DT mice, which will undergo selective ablation of synaptically-connected taste cells, so their role in taste information processing can be directly determined.