Transmission of Taste Signals in Taste Buds

描述（由申请人提供）：味觉受体细胞将化学信号转化为神经信息，传递给支配味蕾的味觉神经纤维。该提案描述了旨在测试假设的实验，即味觉细胞在对刺激做出反应时释放ATP，然后激活味觉神经纤维上的嗜离子嘌呤能受体（P2X2和P2X3）。先前的研究已经证明这两种受体存在于胃内的味觉纤维上。我们的初步数据表明，P2X2/P2X3双敲除小鼠的味觉神经对任何应用于口腔的味觉剂都没有反应，尽管神经对触摸或热刺激有强烈的反应。同样，p2x3 /3 KO小鼠对包括甜味剂在内的许多口味剂和许多苦味物质没有反应，尽管它们对柠檬酸和其他苦味物质有反感反应。这种反应谱与喉上神经相匹配，喉上神经支配着喉上的非味蕾化学感受器。我们提出的实验有三个目的：第一，测试P2X受体对于味觉信息的传递是否必要。对于这些研究，我们将描述系统的行为和电生理学，以测试舌味觉系统的功能程度。我们还将测试喉化学接受是否能解释这些KO小鼠的剩余行为能力。其次，我们将利用解剖学和生理学的方法来测试敲除小鼠的味蕾在结构和功能上是否正常。我们将评估P2X2/3 KO小鼠味蕾的结构和功能，以测试受体细胞是否正常，以及受体上皮是否有明显变化可能导致P2X2/3 KO小鼠味觉功能丧失。在最终目的中，我们将测试野生型和基因敲除小鼠的味蕾是否在刺激时释放ATP。这些功能研究将在体外通过荧光素荧光素酶测定和atp感应生物探针（所谓的“嗅探细胞”）进行。此外，我们将利用阿奎宁负荷来评估味觉细胞对ATP的囊泡吸收和释放。综上所述，这些研究将测试ATP和嗜离子嘌呤能受体在味觉信息传递中的作用。

DESCRIPTION (provided by applicant): Taste receptor cells transduce chemical signals into a neural message transmitted to the gustatory nerve fibers that innervate taste buds. This proposal describes experiments designed to test the hypothesis that in response to stimulation, taste cells release ATP which then activates ionotropic purinergic receptors (P2X2 and P2X3) on the gustatory nerve fibers. Previous studies have demonstrated the presence of these two receptors on intragemmal gustatory fibers. Our preliminary data show that gustatory nerves in P2X2/P2X3 double-knockout mice do not respond to any tastants applied to the oral cavity although the nerves give robust responses to touch or thermal stimuli. Similarly, the P2X2/3 KO mice do not respond behaviorally to many tastants including sweeteners, and many bitter substances although they do respond aversively to citric acid and other bitter substances. This spectrum of responsiveness matches that of the superior laryngeal nerve which innervates non-taste bud chemoreceptors on the larynx. The proposed experiments fall into 3 aims: first to test whether P2X receptors are necessary for transmission of taste information. For these studies we will characterize the system behaviorally and electrophysiologically to test the extent of function of the lingual gustatory system. We will also test whether laryngeal chemoreception accounts for remaining behavioral capabilities of these KO mice. Second, we will utilize anatomical and physiological measures to test whether taste buds in the knockout mice are normal both in terms of structure and function. We will assess structure and function of taste buds in P2X2/3 KO mice to test whether the receptor cells are normal and that no overt changes in the receptor epithelium may underlie the loss of taste function in the P2X2/3 KO mice. In the final aim, we will test whether taste buds in both wildtype and knockout mice release ATP upon stimulation. These functional studies will be carried out in vitro both by using the luciferinluciferase assay and by ATP-sensing biological probes (so-called "sniffer-cells"). In addition, we will utilize quinacrine loading to assess vesicular uptake and release of ATP by taste cells. Taken together, the proposed studies will test the role of ATP and ionotropic purinergic receptors in transmission of taste information.