DIFFERENTIATION OF OLFACTORY RECEPTOR NEURONS IN CULTURE

解剖、分子和功能特性， 哺乳动物嗅觉受体的不同分化阶段 神经元（ORN）还没有被很好地理解。此外， 关于控制系统从一个差异调节运动 阶段到另一个。 为了表征ORN分化阶段， 为了鉴定调控信号，我们开发了分离的细胞培养物 新生和成年大鼠鼻细胞系统。在这些文化中， 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）。 这是迄今为止描述的唯一的分离的文化系统 显示了ORN的成熟。初步数据显示， 表明ORN有几个分化阶段， 神经发生和分化处于单独的调节控制下， 并且特定的球上皮细胞、神经元/神经元接触不是 ORN体外成熟所必需的。 我们在此建议使用这些解离的细胞培养物，在第一个实验中， 这笔赠款的一半，以进一步表征嗅觉的阶段， 神经元分化我们将从解剖学和分子学角度 免疫细胞化学、电镜和原位观察 杂交，与本计划的几个成员合作 项目补助金（PPG）组。我们将描述发展的阶段 的ORN气味反应与电压敏感染料技术， 将这些阶段与分子分化阶段相关联。 在 下半年，我们建议研究具体的监管措施， 控制分化阶段，再次与成员合作 PPG组。特别是，我们建议调查的假设 在培养中，ORN和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， 在增加寿命的ORNs和更多的成熟， 嗅觉标记蛋白阳性的ORN 然后，我们将使用 分离的细胞培养物，以表征特定生长的影响 影响神经元发生和向神经元的初始分化的因素。的 EGF家族生长因子与嗅觉控制有关 神经发生和TGF β家族与最初的 嗅神经元分化这些研究将提供 关于神经元分化的基本机制的信息， 对神经系统的一般功能有指导意义。 此外，获得的关于嗅觉神经元调节的知识 可能适用于其他神经元的发生和分化。 种群的更替和不断的分化， 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.