VOLTAGE DYE CONFOCAL ANALYSIS OF OLFACTORY TRANSDUCTION

嗅觉感受器的气味响应特性研究 神经元（ORN）是困难的，因为微电极记录从一个细胞 并且需要很长一段时间才能为每个细胞提供 不同浓度的气味 因此， 我们知道鼻子是如何区分气味的质量和强度的。 这项建议 使用共焦光学技术对活体中的许多ORN中的每一个进行成像 感觉上皮细胞同时。 细胞膜被染色 电压敏感染料（VSD），当荧光强度发生变化时， 气味刺激细胞。 解决技术问题，包括 检测微小的强度变化，消除 感觉器官，单个细胞和细胞成分的分辨率， 作为纤毛和内部细胞器，减少褪色和毒性的影响 由于光暴露，和可重复的气味传递都得到了解决 在过去的三年里，在这个实验室里。 提出的实验 是因为激光扫描共聚焦显微镜。 这 仪器产生一个非常薄的光学切片通过厚的图像， 组织中 来自切片上方和下方的失焦组织的光， 完全模糊了细胞边界和超微结构， 在共聚焦显微镜中被拒绝。 实验将确定哪些ORN会对多种气味中的每一种做出反应 在低浓度和高浓度下呈现。 得到的响应数组 将描述气味响应选择性，细胞与细胞的相似性，响应 重复性以及失活和恢复动力学。 特别 重要性是对ORN是高度选择性的假设的检验， 也就是说，仅对一种或几种气味表现出高敏感性。 如果是这样的话， 低浓度的气味的呈现应该引起 在数百人的视野中只有几个ORN。 的分数 上皮中每种气味的响应细胞将允许估计 可能需要多种不同的刺激物质， 描述ORN人群。 地形组织的差异 反应细胞的数量将决定 类似的反应细胞。 蛙和蝾螈的嗅上皮 因为过去获得的数据表明， 物种在气味转导和化学定位方面不同， organization. 将在存在正常粘液的情况下测量反应， 用粘液耗尽来测试气味传导需要 一种细胞外辅助因子 VSD已被识别， 染色嗅上皮中的支持细胞（SC）。 这些细胞 被认为影响嗅觉传导。 程度和选择性 将确定SC中气味诱发的膜电位变化，以测试 这个假设。 VSD和共聚焦显微镜具有独特的能力， 解决气味检测和编码中的主要问题， 微电极生理学的实验研究。 嗅觉在调节体内激素状态方面起着重要作用， 情绪倾向饥饿和社会行为 这里提出的工作 将促进对细胞过程的理解， 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.