Engele J, Pilgrim C, Kirsch M, Reisert I
Abteilung Anatomie und Zellbiologie, Universität Ulm, F.R.G.
Brain Res. 1989 Mar 27;483(1):98-109. doi: 10.1016/0006-8993(89)90039-5.
Morphological and functional differentiation of dopamine (DA) neurons was compared in dissociated cultures from gestational day 14 rat mesencephalon and diencephalon. Numbers of tyrosine hydroxylase-immunoreactive (TH-IR) neurons relative to all neurons were 4 and 1.7 times higher in mesencephalic than in diencephalic cultures at 6 and 13 days in vitro (DIV), respectively. Morphological maturation of diencephalic DA neurons was retarded in comparison to mesencephalic DA neurons. There were gross differences in soma size and length of processes between the two types of DA neurons, the appearance of which was strongly reminiscent of DA cell types described in vivo. Functional maturation of DA neurons was quantified by measuring uptake and Ca2+-dependent K+-stimulated release of [3H]DA per TH-IR neuron. As early as 6 DIV, DA uptake by mesencephalic DA neurons was saturable, was sensitive to benztropine and reserpine, and could be displaced by unlabeled DA. Twenty to 30% of the radioactivity accumulated could be released upon depolarization within a period of 5 min. At 6 DIV, influx of [3H]DA into diencephalic DA neurons was almost insensitive to benztropine, reserpine and unlabeled DA. Even after 13 DIV, diencephalic DA uptake was characterized by a markedly lower initial velocity, a longer time needed to reach saturation, a lower uptake capacity, and a lower sensitivity to benztropine than mesencephalic DA uptake. The releasable pool was very small and did not increase between DIV 6 and 13. The results demonstrate that mesencephalic DA neurons in vitro differentiate considerably faster than diencephalic DA neurons and gain functional competence very early in brain development. Comparison with data on adult nigrostriatal and hypothalamic DA systems suggests that the in vitro differences reflect a fundamental regional diversity of DA neurons.
对妊娠第14天大鼠中脑和间脑分离培养物中多巴胺（DA）神经元的形态和功能分化进行了比较。在体外培养6天和13天时，中脑培养物中酪氨酸羟化酶免疫反应性（TH-IR）神经元相对于所有神经元的数量分别比间脑培养物中的高4倍和1.7倍。与中脑DA神经元相比，间脑DA神经元的形态成熟延迟。两种类型的DA神经元在细胞体大小和突起长度上存在显著差异，其外观强烈让人联想到体内描述的DA细胞类型。通过测量每个TH-IR神经元对[3H]DA的摄取和Ca2+依赖性K+刺激释放来量化DA神经元的功能成熟。早在体外培养6天时，中脑DA神经元对DA的摄取是可饱和的，对苯海索和利血平敏感，并且可以被未标记的DA取代。在5分钟内去极化时，积累的放射性中有20%至30%可以释放。在体外培养6天时，[3H]DA进入间脑DA神经元的流入对苯海索、利血平及未标记的DA几乎不敏感。即使在体外培养13天后，间脑DA摄取的特点仍然是初始速度明显较低、达到饱和所需时间更长、摄取能力较低以及对苯海索的敏感性低于中脑DA摄取。可释放池非常小，在体外培养6天至13天之间没有增加。结果表明，体外培养的中脑DA神经元比间脑DA神经元分化得快得多，并且在脑发育的早期就获得了功能能力。与成年黑质纹状体和下丘脑DA系统的数据比较表明，体外差异反映了DA神经元的基本区域多样性。
