VOLTAGE DYE CONFOCAL ANALYSIS OF OLFACTORY TRANSDUCTION

嗅觉感受器嗅觉反应特性的研究 神经元(ON)是困难的，因为微电极记录来自一个细胞 并且需要一段较长的时间来呈现给每个细胞 几种不同浓度的气味。因此，它不是 知道鼻子如何区分气味的质量和强度。这项建议 使用共焦光学技术对活着的许多兽人中的每一个进行成像 同时还有感觉上皮细胞。细胞膜被染上 电压敏感染料(VSD)在以下情况下会改变荧光强度 气味会刺激细胞。具有挑战性的技术问题包括 检测到微小的强度变化，消除了运动的 感觉器官，单个细胞和细胞成分的分辨率，如 作为纤毛和内部细胞器，最大限度地减少褪色和毒性的影响 由于光照和可重复的气味传递，所有问题都已解决 在过去的三年里在这个实验室里。建议进行的实验 因为有了激光扫描共聚焦显微镜才有可能。这 仪器产生一个非常薄的光学切片穿过厚度的图像 纸巾。来自该部分上方和下方的散焦组织的光，其 完全模糊了传统的细胞边界和超微结构 荧光显微镜，是在共聚焦显微镜中被拒绝的。 实验将确定哪一种鸟对多种气味有反应 以低浓度和高浓度呈现。生成的响应数组 将描述气味响应选择性、细胞间相似性、响应 重复性，以及失活和恢复动力学。特别的 重要性是对兽类具有高度选择性的假设的检验， 即，只对一种或几种气味表现出高敏感性。如果是的话， 在低浓度下呈现气味应该会在 只有几个兽人在数百人的视野中。小数部分 上皮中每种气味的反应细胞将允许估计 可能需要的不同刺激物质的数量 描述ORN群体的特征。地形组织的差异 反应细胞的数量将决定解剖聚集的程度 反应相似的细胞。青蛙和火蜥蜴的嗅觉上皮 会被比较，因为过去获得的数据表明，这些 在气味转导和化学作用方面，物种是不同的 组织。反应将在正常粘液存在的情况下进行测量 随着粘液的耗尽来检验气味转导需要 一种细胞外辅助因子。VSD已被识别为选择性地 嗅觉上皮支持细胞(SCs)染色。这些细胞是 据推测会影响嗅觉传导。范围和选择性 气味诱发的SCs的膜电位变化将被确定为测试 这一假设。VSD和共聚焦显微镜是唯一能够 解决气味检测和编码中的主要问题 用微电极生理学进行实验研究。 嗅觉在调节身体荷尔蒙状态方面起着重要作用， 情绪倾向、饥饿和社会行为。在这里提出的工作 将促进对辅助的细胞过程的理解 嗅觉功能。

Investigations of the odor-response properties of olfactory receptor neurons (ORNs) are difficult because a microelectrode records from one cell at a time and it takes an extended period of time to present each cell with many odors at several different concentrations. As a result it is not known how the nose distinguishes odor quality and intensity. This proposal uses confocal optical technology to image each of many ORNs in the living sensory epithelium simultaneously. The cell membranes are stained with voltage-sensitive dyes (VSDs) which change in fluorescence intensity when an odor stimulates the cells. Challenging technical problems including detection of small intensity changes, elimination of movement of the sensory organ, resolution of individual cells and of cell components such as cilia and internal organelles, minimizing effects of fading and toxicity due to light exposure, and repeatable odor delivery have all been resolved in this laboratory during the past three years. The experiments proposed are possible because of the laser scanning confocal microscope. This instrument produces an image of a very thin optical slice through thick tissues. Light from out-of-focus tissue above and below the section, which completely obscures cell boundaries and ultrastructure in the conventional epifluorescence microscope, is rejected in the confocal microscope. Experiments will determine which ORNs respond to each of many odors presented at low and high concentrations. The resulting response array will describe odor response selectivity, cell-to-cell similarity, response repeatability, and inactivation and recovery kinetics. Of particular importance is a test of the hypothesis that ORNs are highly selective, i.e., show high sensitivity to only one or a few odors. If so, presentation of an odor at low concentrations should evoke responses in only a few ORNs in the visual field of many hundred. The fraction of responding cells per odor in the epithelium will allow an estimate of the number of different stimulus substances likely to be required to characterize the ORN population. Differences in topographical organization of responding cells will determine the extent of anatomical clustering of similarily responding cells. Olfactory epithelia of frogs and salamanders will be compared because data obtained in the past suggests that these species are different with respect to odor transduction and chemotopic organization. Responses will be measured with normal mucus present and with mucus depleted to test the hypothesis that odor transduction requires an extracellular co-factor. VSDs have been identified which selectively stain supporting cells (SCs) in the olfactory epithelium. These cells are postulated to influence olfactory transduction. The extent and selectivity of odor-evoked membrane potential changes in SCs will be determined to test this postulate. VSDs and confocal microscopy are uniquely capable of resolving major issues in odor detection and coding which are not amenable to experimental study with microelectrode physiology. The olfactory sense has a major role in regulating body hormonal state, emotional disposition, hunger, and social behavior. The work proposed here will advance understanding of the cellular processes which subserve olfactory sensory function.