VOLTAGE CLAMP PROBE OF TASTE RESPONSES IN DEVELOPMENT

描述：（改编自申请人摘要）。味觉刺激与味蕾中的味觉敏感细胞相互作用，其中大多数位于舌头上表面的乳头中。具有食欲的物质（例如，甜的刺激物）通常被摄取;那些令人厌恶的刺激物（例如，痛苦的刺激）被拒绝，尽管这些倾向可以通过经验来修改。味觉偏好发生的一些变化似乎与年龄有关，这在啮齿动物模型中得到了很好的证明。在出生后的早期发育中，对氯化钠的味觉敏感性的变化尤其明显。对氯化钠的敏感性在出生时就存在，但后来发展得更多，特别是当动物断奶时。有两种模式的咸味感知，这似乎驻留在不同的区域的味蕾。味觉细胞顶端微绒毛上的一个传感器与口腔液体直接接触。另一种被隔离在细胞的基底外侧膜上，使得一些刺激必须通过将细胞的顶侧域和基底外侧域分开的紧密连接扩散以到达受体位点。数据表明，访问这些基底外侧网站可以被钙阻断，并且在发育过程中，味觉刺激的紧密连接渗透性发生变化。味觉神经反应的体内记录，与电压钳下的感受野，将用于筛选候选刺激的基底外侧受体位点在发育和成熟的大鼠。与此同时，一种新开发的体外技术将被用来直接测量跨单个菌状乳头的细胞旁区域的各种刺激的通量。荧光染料和离子选择性微电极都将用于测量由于几种酸、各种钠盐和钾盐、钙离子和铵离子衍生物而产生的氢离子通量。钙是特别重要的，因为它预计将减少其自身的细胞旁渗透性以及其他刺激。这些实验将确定味蕾基底外侧刺激物的刺激浓度的实际范围，初步结果表明其远低于放置在舌头上的浓度。还将开发方法来确定氢和钙的侧向细胞间隙的缓冲特性。

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.