Heller A, Kontur P, Hoffmann P, Heller B
University of Chicago, Department of Pharmacological and Physiological Sciences, IL 60637.
J Neurosci Methods. 1988 Aug;25(1):83-90. doi: 10.1016/0165-0270(88)90123-9.
The study of cellular interactions during the development of the nervous system is facilitated by the use of the reaggregate tissue culture system. In this system, embryonic neurons are dissociated from one another and allowed to reaggregate in rotatory culture, where they resume their normal differentiation. Reaggregates provide a model for studying neuronal development and cell interactions under conditions which allow for the control of a large number of variables, such as the numbers of cells which are allowed to interact, the presence or absence of appropriate target cells, and the chemical environment in which the neurons develop. For quantitative purposes, it is necessary to estimate the numbers of cells of a given neurochemical or morphologic type. Here we describe a computer-assisted counting system which allows one to determine the numbers of neurochemically identified neurons within reaggregate cultures from the counting of such neurons in a randomly selected sample of thirty 10-micron sections from the reaggregates in a given flask. These sections represent less than 0.5% of the total number of sections derived from the reaggregates in a single flask. In addition, the system allows one to quantitatively compare differences in the numbers of such neurons between experimental flasks utilizing appropriate statistical methods. A comparison of the number of central dopaminergic neurons estimated by this method with the direct counting of such neurons in serial sections of single reaggregates resulted in excellent agreement as to the number of dopaminergic neurons within a given experimental flask.(ABSTRACT TRUNCATED AT 250 WORDS)
利用再聚合组织培养系统有助于对神经系统发育过程中的细胞相互作用进行研究。在这个系统中，胚胎神经元彼此分离，并在旋转培养中重新聚集，在那里它们恢复正常分化。再聚集体为研究神经元发育和细胞相互作用提供了一个模型，在此条件下可以控制大量变量，例如相互作用的细胞数量、是否存在合适的靶细胞以及神经元发育所处的化学环境。出于定量目的，有必要估计给定神经化学或形态学类型的细胞数量。在这里，我们描述了一种计算机辅助计数系统，该系统允许通过对来自给定培养瓶中再聚集体的三十个10微米随机选择切片样本中的此类神经元进行计数，来确定再聚合培养物中神经化学鉴定的神经元数量。这些切片占单个培养瓶中再聚集体衍生切片总数的不到0.5%。此外，该系统允许使用适当的统计方法对实验培养瓶之间此类神经元数量的差异进行定量比较。通过这种方法估计的中枢多巴胺能神经元数量与在单个再聚集体连续切片中对这些神经元的直接计数相比较，在给定实验培养瓶中多巴胺能神经元数量方面达成了极好的一致性。（摘要截短至250字）
