DIFFERENTIATION OF OLFACTORY RECEPTOR NEURONS IN CULTURE

解剖、分子和功能特性 哺乳动物嗅觉感受器分化的不同阶段 对神经元(Orns)的了解还不是很清楚。此外，人们对此知之甚少 关于运动调控系统的一种辨析 到另一个舞台。为了表征ORN分化阶段和 识别调控信号，我们已经开发出分离的细胞培养 新生和成年大鼠鼻腔细胞系统。在这些文化中，兽人是 产生并经历初始分化和成熟 (对气味的电反应和嗅觉标记的表达 蛋白质)。这是迄今为止所描述的唯一分离的培养系统。 这表明了奥恩斯的成熟。我们的初步数据显示或 表明鸟巢有几个分化阶段， 神经发生和分化受到单独的监管控制， 而特定的球上皮细胞、神经元/神经元接触并不是 是ORN体外成熟所必需的。 我们在这里建议使用这些分离的细胞培养物，在第一 这笔拨款的一半，以进一步描述嗅觉的各个阶段 神经元分化。我们将分析解剖学和分子学 应用免疫细胞化学、电子显微镜和原位技术观察ONS的变化 杂交，与本计划的几个成员合作 项目赠款(PPG)组。我们将描述开发过程中的各个阶段 ORN气味反应与电压敏感染料技术和 将这些阶段与分子分化的阶段联系起来。在 在这笔拨款的后半部分，我们建议调查特定的监管机构 对分化阶段的控制，再次与成员合作 PPG组的成员。特别是，我们建议对这一假设进行调查 培养中Orns与嗅球神经元之间的神经元接触 将为Orns提供额外的营养支持，从而 在鸟的寿命的增加和更多的成熟鸟中， 嗅觉标记，蛋白质阳性的鹰嘴鸟。然后，我们将使用 分离的细胞培养以表征特定生长的影响 影响神经元发生和向神经元分化的因素。这个 EGF家族生长因子与嗅觉控制有关 神经发生和转化生长因子β家族参与了早期的 嗅神经细胞分化。这些研究将提供 关于神经元分化的基本机制的信息将是 对神经系统的总体功能有指导意义的。 此外，还获得了有关嗅觉神经元调节的知识 发生和分化可能适用于其他神经元 更替和持续分化的种群 神经元的成熟不会发生。

The anatomical, molecular and functional properties that characterize different stages of differentiation of the mammalian olfactory receptor neurons (ORNs) are not well understood. In addition, little is known about the control systems regulating movement from one differentiation stage to another. To characterize ORN differentiation stages and identify regulatory signals, we have developed dissociated cell culture systems of newborn and adult rat nasal cells. In these cultures, ORNs are generated and undergo both initial differentiation and maturation (electrical responses to odorants and expression of the olfactory marker protein). This is the only dissociated culture system described to date that shows maturation of ORNs. Our preliminary data have shown or suggested that ORNs have several stages of differentiation, that neurogenesis and differentiation are under separate regulatory control, and that specific bulb-epithelial, neuron/neuron contacts are not necessary for ORN maturation in vitro. We propose herein to use these dissociated cell cultures, in the first half of this grant, to further characterize the stages of olfactory neuronal differentiation. We will analyze anatomical and molecular changes in ORNs with immunocytochemistry, electron microscopy and in situ hybridization, in collaborations with several members of this Program Project Grant (PPG) group. We will characterize stages in the development of ORN odorant responses with the voltage sensitive dye technology and correlate these stages with stages in molecular differentiation. In the second half of this grant, we propose to investigate specific regulatory controls of differentiation stages, again in collaboration with members of the PPG group. In particular, we propose to investigate the hypothesis that neuronal contact between ORNs and olfactory bulb neurons in culture will provide an additional level of trophic support to ORNs, resulting in an increase in the life span of ORNs and greater numbers of mature, olfactory marker, protein-positive ORNs. Then, we will use the dissociated cell cultures to characterize the effects of specific growth factors on neuronal genesis and initial differentiation into neurons. The EGF family of growth factors has been implicated in control of olfactory neurogenesis and the TGFbeta family has been implicated in initial olfactory neuronal differentiation. These studies will provide information on basic mechanisms of neuronal differentiation that will be instructive of the function of the nervous system in general. Furthermore, knowledge gained about the regulation of olfactory neuronal generation and differentiation might be applicable to other neuronal populations where replacement and continual differentiation and maturation of neurons do not occur.