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，大部分味蕾位于舌头上表面的乳头状细胞中。通常摄入具有开胃味道的物质（如甜味刺激）；那些令人厌恶的（例如，苦涩的刺激）被拒绝，尽管这些倾向可以通过经验来改变。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得到了充分的证明。味觉对氯化钠的敏感性随出生后早期发育的变化尤其明显。出生时对氯化钠有一定的敏感性，但后来就会发展起来，特别是在断奶后。有两种盐味感知模式，它们似乎位于味蕾的不同区域。味觉细胞顶端微绒毛上的一个传感器直接与口腔液体接触。另一种被隔离在细胞的基底外侧膜上，因此一些刺激必须通过分离细胞顶端和基底外侧结构域的紧密连接扩散到受体部位。数据表明，进入这些基底外侧部位的通道可以被钙阻断，并且在发育过程中，对味觉刺激的紧密连接通透性会发生变化。在体内记录的味觉神经反应，与接受野在电压钳下，将用于筛选候选刺激的基底外侧受体位置在发育和成熟的大鼠。同时，一项新开发的体外技术将用于直接测量各种刺激在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性微电极都将用于测量由于几种酸、各种钠和钾盐、钙离子和铵离子衍生物引起的氢离子通量。钙是特别重要的，因为它预计会降低其自身的细胞旁通透性以及其他刺激。这些实验将确定味蕾基底外侧的刺激浓度的实际范围，初步结果表明，其浓度远低于舌头上的浓度。还将开发方法来确定氢和钙的横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.