SEX HORMONES AND THE SINGLE NEURON

导论. 性腺类固醇激素发挥了深刻的 对下丘脑的影响 然而，性激素的作用 在单个神经元上不容易在体内研究， 完整（整合）中枢神经系统的复杂性， 难以控制变量。 为了避免这些问题， 细胞培养用于阐明神经调控事件，但 由于无法获得分离的神经元， 的定量数据。 具体目标和方法。 近日 显示，当在三维（3-D） 细胞外基质（ECM），单个下丘脑神经元不参与 与任何其他类型的细胞接触， 可辨认的轴突和树突。 生长和分化 这些过程可以用绝对值可靠地量化， 相对而言。 这种新方法将用于：1。确定 性类固醇、17 B雌二醇、睾酮和 双氢睾酮对单细胞生长和分化的影响 下丘脑神经元的三维ECM。 2.确定是否促黄体生成 激素释放激素（LHRH）、加压素（VP）和催产素 (OXY)可以在单个下丘脑神经元中证明， 无论是培养条件还是影响肽的因素 释放将改变合成-分泌。 图像分析系统 与相衬显微镜和微型计算机相连接， 用于衡量下列增长指数， 随时间的分化：轴突长度（单位：μ m/神经元）;树突 长度（单位：um/神经元）;所有突起的总合并长度（单位：um/神经元） um/神经元;初级突起，分支点， 节段和终末/神经元。 Student t检验或Anova检验 用于比较治疗组之间的数据。 光和 电子显微镜将用于定性评价 cultures. 免疫细胞化学和放射免疫分析将 用于评估LHRH、VP和OXY。 意义 治疗 并且没有性腺类固醇或影响肽释放的因素 可以在发育中的单个下丘脑的培养物中进行研究 神经元 结果应提供对反馈或其他 在细胞水平上，调节生长和 下丘脑神经元的分化或分泌 用于繁殖。

Introduction. Gonadal steroid hormones exert a profound influence on the hypothalamus. However, actions of sex hormones on single neurons are not easily studied in vivo due to the complexity of the intact (integrated) central nervous system and difficulty in controlling variables. To circumvent these problems, cell culture is used to elucidate hormonally regulated events, but is limited by the inability to obtain isolated neurons and the lack of quantitative data. Specific Aims and Methods. Recently, it was shown that when cultured in a three-dimensional (3-D) extracellular matrix (ECM), single hypothalamic neurons not in contact with any other cell type rapidly grow morphologically identifiable axons and dendrites. Growth and differentiation of these processes can be reliably quantified in absolute rather than relative terms. This new method will be used to: 1. Determine the effects of the sex steroids, 17 B estradiol, testosterone and dihydrotestosterone, on growth and differentiation of single hypothalamic neurons in a 3-D ECM. 2. Determine if luteinizing hormone releasing hormone (LHRH), vasopressin (VP) and oxytocin (OXY) can be demonstrated in single hypothalamic neurons and whether culture conditions or factors that influence peptide release will alter synthesis-secretion. An image analysis system coupled to a phase contrast microscope and microcomputer will be used to measure the following indices of growth and differentiation over time: axon length in um/neuron; dendrite length in um/neuron; total combined length of all processes in um/neuron; the number of primary processes, branch points, segments and terminals/neuron. A Student's t or Anova test will be used to compare data between treatment groups. Light and electron microscopy will be used to qualitatively evaluate the cultures. Immunocytochemistry and radioimmunoassay will be used to assess LHRH, VP and OXY. Significance. Treatment with and without gonadal steroids or factors affecting peptide release can be investigated in cultures of developing single hypothalamic neurons. Results should provide insight into feedback or other mechanisms, at the cellular level, that regulate growth and differentiation or secretion of hypothalamic neurons important for reproduction.