Christie D S, Forbes M E, Maxwell G D
Department of Anatomy, University of Connecticut Health Center, Farmington 06032.
J Neurosci. 1987 Nov;7(11):3749-63. doi: 10.1523/JNEUROSCI.07-11-03749.1987.
We have investigated several phenotypic features of the catecholamine-positive (CA+) cell population that develops in quail neural crest cultures. The number, spatial distribution, and morphology of CA+ and tyrosine hydroxylase-positive (TH+) cells are similar at all ages examined, suggesting that these 2 cell classes are identical. Neither CA+ nor TH+ cell bodies or processes were stained using antisera that recognize the 70 or 160 kDa subunits of chicken neurofilament protein. Other cell bodies and fibers in the cultures (which were CA- and TH-) were stained with these neurofilament antisera. The uptake and storage of 3H-norepinephrine by neural crest cultures containing CA+ cells were inhibited in the presence of desmethylimipramine and by incubation at 0 degrees C, but were unaffected by normetanephrine. Overnight treatment with reserpine eliminated histochemically detectable CA fluorescence from the cultures. Chronic reserpine treatment from day 2 to 7 in vitro prevented the appearance of CA+ cells, while normal numbers of TH+ and somatostatin-like immunoreactive (SLI) cells developed. The number and light-microscopic morphology of the CA+ cells that developed in these cultures were not dramatically altered by either exogenous NGF or 6-hydroxydopamine. Using the method of Grillo et al. (1974), we have demonstrated that the CA+ cells observed in the light microscope corresponded to cells containing abundant cytoplasmic granular vesicles (GV) characteristic of catecholamine storage granules observed in other systems. The GV diameters were quite similar in cells examined after 5, 7, 14, and 21 d in vitro. Most GV were 50-200 nm in diameter and were distributed in a unimodal manner, with the observed modal values in the range of 85-115 nm at the ages examined. The number of GV/micron2 of cytoplasmic area remained quite constant at all ages examined. These data, taken together with other available information, suggest that the CA+ cells that differentiate in our neural crest cultures resemble, in many respects, the small, intensely fluorescent cells found in autonomic ganglia and extra-adrenal chromaffin tissue of many species. At present, we do not know if the CA+ cells that differentiate in our neural crest cultures are a stable endpoint of development or whether they are a developmental intermediate in adrenergic differentiation that is normally observed only transiently during the development of avian sympathetic ganglia in vivo, but that can persist under our tissue culture conditions.
我们研究了鹌鹑神经嵴培养物中发育的儿茶酚胺阳性（CA+）细胞群体的几个表型特征。在所有检测的年龄阶段，CA+细胞和酪氨酸羟化酶阳性（TH+）细胞的数量、空间分布及形态均相似，这表明这两类细胞是相同的。使用识别鸡神经丝蛋白70或160 kDa亚基的抗血清，未对CA+或TH+细胞体及突起进行染色。培养物中的其他细胞体和纤维（即CA-和TH-细胞）被这些神经丝抗血清染色。在去甲丙咪嗪存在的情况下以及在0℃孵育时，含有CA+细胞的神经嵴培养物对3H-去甲肾上腺素的摄取和储存受到抑制，但去甲变肾上腺素对其无影响。利血平过夜处理消除了培养物中组织化学可检测到的CA荧光。在体外从第2天至第7天进行慢性利血平处理可阻止CA+细胞的出现，而TH+细胞和生长抑素样免疫反应性（SLI）细胞数量正常发育。在这些培养物中发育的CA+细胞的数量和光学显微镜形态，在外源性NGF或6-羟基多巴胺作用下均未发生显著改变。使用Grillo等人（1974年）的方法，我们已证明在光学显微镜下观察到的CA+细胞对应于含有丰富细胞质颗粒小泡（GV）的细胞，这些小泡具有在其他系统中观察到的儿茶酚胺储存颗粒的特征。在体外培养5、7、14和21天后检测的细胞中，GV直径非常相似。大多数GV直径为50 - 200 nm，呈单峰分布，在所检测的年龄阶段，观察到的众数在85 - 115 nm范围内。在所有检测的年龄阶段，每平方微米细胞质区域的GV数量保持相当恒定。这些数据与其他现有信息一起表明，在我们的神经嵴培养物中分化的CA+细胞在许多方面类似于在许多物种的自主神经节和肾上腺外嗜铬组织中发现的小而强荧光细胞。目前，我们不知道在我们的神经嵴培养物中分化的CA+细胞是发育的稳定终点，还是它们是肾上腺素能分化过程中的发育中间体，这种中间体在体内禽类交感神经节发育过程中通常仅短暂出现，但在我们的组织培养条件下可以持续存在。
