Fiszman M L, Behar T, Lange G D, Smith S V, Novotny E A, Barker J L
Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.
Brain Res Dev Brain Res. 1993 Jun 8;73(2):243-51. doi: 10.1016/0165-3806(93)90144-y.
Single cell suspensions derived from embryonic telencephala taken from embryos of gestational day 13 (E13) as well as rat striatal tissue from E14, 15 and 17 were prepared by tissue digestion with papain. Cell suspensions were analyzed by flow cytometry or plated onto poly-D-lysine-coated culture dishes for either nuclear staining or immunocytochemistry. Experiments on functional Na+ channels and GABAA receptor expression were carried out using a fluorescence-activated cell sorter (FACS) and a negatively charged fluorescent indicator dye (oxonol). FACS analysis of embryonic cell suspensions at E13-17 consistently revealed one major subpopulation accounting for 85-90% of the events and one minor subpopulation (10-15% of the total). When sorted, the major subpopulation consisted of phase-bright cells of 5-7 microns diameter some of which had neurites. The minor population consisted of phase-dark cells and resealed membranes of 0.5-4 microns diameter as well as debris. Almost all the cells obtained in the high FALS (forward-angle light scatter) subpopulation at E17 expressed 200-kDa neurofilament and tetanus toxin antigens while the small diameter cells seldom expressed tetanus toxin and particles never did. A small number of GABA-containing neurons were detected in the telencephalon at E13 (3%) and in the developing striatum at E14 (6%). All of the GABA-containing neurons expressed neurofilament. In the embryonic rat striatum, nanomolar concentrations of muscimol (GABAA agonist) induced depolarizing responses. A small number of cells in the high FALS subpopulation were responsive to muscimol starting at embryonic day 14, and the number of responsive cells increased at E15.(ABSTRACT TRUNCATED AT 250 WORDS)
从妊娠第13天(E13)胚胎的端脑以及E14、E15和E17大鼠纹状体组织中获取的单细胞悬液,通过木瓜蛋白酶消化组织来制备。细胞悬液通过流式细胞术进行分析,或接种到聚-D-赖氨酸包被的培养皿上用于细胞核染色或免疫细胞化学。使用荧光激活细胞分选仪(FACS)和带负电荷的荧光指示剂染料(氧杂菁)进行功能性钠离子通道和GABAA受体表达的实验。对E13 - 17胚胎细胞悬液的FACS分析始终显示一个主要亚群占事件的85 - 90%,以及一个次要亚群(占总数的10 - 15%)。分选后,主要亚群由直径为5 - 7微米的亮相细胞组成,其中一些有神经突。次要群体由直径为0.5 - 4微米的暗相细胞和重新封闭的膜以及碎片组成。在E17的高FALS(前向角光散射)亚群中获得的几乎所有细胞都表达200 kDa神经丝和破伤风毒素抗原,而小直径细胞很少表达破伤风毒素,颗粒则从不表达。在E13时,端脑中检测到少量含GABA的神经元(3%),在E14时发育中的纹状体中检测到少量含GABA的神经元(6%)。所有含GABA的神经元都表达神经丝。在胚胎大鼠纹状体中,纳摩尔浓度的蝇蕈醇(GABAA激动剂)诱导去极化反应。从胚胎第14天开始,高FALS亚群中的少量细胞对蝇蕈醇有反应,在E15时反应细胞数量增加。(摘要截于250字)