VOLTAGE CLAMP PROBE OF TASTE RESPONSES IN DEVELOPMENT

描述：(改编自申请人的摘要)。味觉刺激与味蕾中的味觉敏感细胞相互作用，其中大部分位于舌头上表面的乳头中。有食欲的物质(如甜味刺激)通常会被摄入；那些令人厌恶的物质(如苦味刺激)则会被拒绝，尽管这些倾向可以被经验改变。味觉偏好的一些变化似乎与年龄有关，这在啮齿动物模型中已经得到了很好的证明。随着出生后的早期发育，对氯化钠的味觉敏感性的变化尤其明显。在出生时对氯化钠有一定的敏感性，但在以后，特别是在动物断奶的时候，会有很大的发展。有两种盐味感知模式，它们似乎位于味蕾的不同区域。一个传感器位于味觉细胞的顶端微绒毛上，与口腔液体直接接触。另一种是隔离在细胞的基侧膜上，因此一些刺激必须通过分隔细胞顶端和基侧区域的紧密连接扩散，才能到达受体位置。数据表明，进入这些基底外侧部位的通道可以被钙阻断，而且在发育过程中，对味觉刺激的紧密连接渗透性会发生变化。味觉神经反应的活体记录，以及电压钳下的感受野，将用于筛选发育中和成熟大鼠的基底外侧感受器位置的候选刺激。同时，一项新开发的体外技术将被用来直接测量不同刺激跨单个真菌状乳头的细胞旁区域的通量。荧光染料和离子选择微电极都将被用来测量几种酸、各种钠盐和钾盐、钙离子和氨离子衍生物的氢离子通量。钙尤其重要，因为它有望降低自身的细胞旁通透性以及其他刺激的通透性。这些实验将确定味蕾基底外侧刺激的实际浓度范围，初步结果表明，刺激浓度远远低于舌头上的浓度。还将开发确定横向细胞间隙对氢和钙的缓冲性能的方法。

DESCRIPTION: (Adapted from the Applicant's Abstract). Taste stimuli interact with taste-sensitive cells in taste buds, the majority of which are located in papillae on the upper surface of the tongue. Substances with appetitive tastes (e.g., sweet stimuli) are normally ingested; those that are aversive (e.g., bitter stimuli) are rejected, although these tendencies can be modified by experience. Some of the changes that occur in taste preferences appear to be age related and this has been well-documented in rodent models. Changes in taste sensitivity to sodium chloride with early postnatal development are especially clear. Some sensitivity to sodium chloride is present at birth, but much develops later, particularly when the animals are weaned. There are two modes of salt-taste sensing, which appear to reside in different regions of the taste bud. One sensor, on the apical microvilli of taste cells, makes direct contact with the mouth fluid. The other is sequestered on the basolateral membrane of the cells so that some stimuli must diffuse through the tight junctions separating the apical and basolateral domains of the cells to reach the receptor sites. Data suggest that access to these basolateral sites can be blocked by calcium, and that the tight junction permeability to taste stimuli changes during development. In vivo recordings of taste nerve responses, with the receptive field under voltage-clamp, will be used to screen for candidate stimuli of the basolateral receptor sites in developing and mature rats. In parallel, a newly developed in vitro technique will be used to make direct measurements of fluxes of various stimuli across the paracellular regions of single fungiform papillae. Both fluorescent dyes and ion selective microelectrodes will be used to measure fluxes of hydrogen ions due to several acids, various salts of sodium and potassium, calcium ions, and ammonium ion derivatives. Calcium is especially important because it is expected to reduce its own paracellular permeability as well as that of other stimuli. These experiments will determine the actual range of stimulating concentrations of stimuli on the basolateral side of the taste buds, which preliminary results indicate are far lower than the concentrations placed on the tongue. Methods will also be developed to determine the buffering properties of the lateral intercellular spaces for hydrogen and calcium.