SEX HORMONES AND THE SINGLE NEURON

导言。性腺类固醇激素对 对下丘脑的影响。然而，性激素的作用 对单个神经元的研究不容易在体内进行，因为 完整(完整)的中枢神经系统的复杂性和 难以控制变量。为了绕过这些问题， 细胞培养被用来阐明激素调节事件，但 受限于无法获得分离的神经元和缺乏 量化数据。具体目标和方法。最近，它 研究表明，当在三维(3-D)培养时 细胞外基质(ECM)，单个下丘脑神经元不在 与任何其他细胞类型的接触在形态上迅速增长 可辨认的轴突和树突。的成长和分化 这些过程可以可靠地量化为绝对值，而不是 相对术语。这种新方法将用于：1.确定 性激素、17-B雌二醇、睾酮和 双氢睾酮对单细胞生长分化的影响 3-D ECM中的下丘脑神经元。2.确定黄体生成素是否 激素释放激素(LHRH)、加压素(VP)和催产素 (Oxy)可在单个下丘脑神经元和 无论是培养条件还是影响多肽的因素 释放会改变合成和分泌。一种图像分析系统 耦合到相差显微镜和微型计算机将 用于衡量以下增长指数和 随时间的分化：微米/神经元中的轴突长度；树突 以微米/神经元为单位的长度；所有突起的总总长度 UM/神经元；初级突起、分支点、 节段和终末/神经元。学生t检验或方差检验将 用于比较治疗组之间的数据。光和 将使用电子显微镜来定性评估 文化。免疫细胞化学和放射免疫分析将 用于评估LHRH、VP和Oxy。意义重大。通过以下方式治疗 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中进行研究 神经元。结果应提供对反馈或其他方面的洞察 在细胞水平上，调节生长和 下丘脑神经元分化或分泌的重要作用 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.