鸡胚视网膜纯化神经元和非神经元培养物中γ-氨基丁酸（GABA）的摄取与释放

Hyndman A G, Adler R

Brain Res. 1982 Feb;255(2):167-80. doi: 10.1016/0165-3806(82)90018-9.

Uptake and release of gamma-aminobutyric acid (GABA) have been studied using glia-free, purified neuronal cultures from 8-day chick embryo retina. At 3 days in vitro 65% of the neurons showed high-affinity GABA uptake. These neurons appeared heavily labeled after incubation in 5 X 10(-8) M [3H]GABA, but no labeling was detected when the incubation was carried out at 4 degrees C, or in the absence of Na+ ions. Diaminobutyric acid (DABA) also blocked completely the neuronal uptake of GABA, while beta -alanine was ineffective at similar concentrations. At 6 days in vitro Na+- and temperature-dependent GABA uptake was present in 50% of the neurons. In addition, in 80% of those neurons the uptake was insensitive to DABA or beta -alanine, whereas in the remaining 20% it was blocked by DABA but not by beta -alanine. Important developmental changes were also found in the capacity of the neurons to release GABA into the medium. Spontaneous GABA release (i.e. that taking place in regular medium, containing 5 mM K+) was higher at 3 than at 6 days in vitro. However, increasing the K+ concentration to 56 mM had minimal effects at 3 days in vitro, but induced a 2 to 3-fold increase in GABA release at 6 days in vitro. This K+-induced release appeared to be Ca2+-dependent, since it was substantially reduced the presence of 10 mM Co2+. Cultures containing a confluent monolayer of nonneuronal flat cells were generated by seeding retinal cell suspensions on poorly adhesive substrata. Retina nonneuronal cells showed, during the first 10 days in vitro, a high-affinity mechanism for GABA uptake which was Na+- and temperature-dependent, and was reduced by 85% by DABA but was practically unaffected by beta-alanine. This uptake mechanism seemed to be lost towards the end of the second week in vitro, and could not be detected after 21 days culture.

利用8日龄鸡胚视网膜的无胶质、纯化神经元培养物，对γ-氨基丁酸（GABA）的摄取和释放进行了研究。在体外培养3天时，65%的神经元表现出高亲和力的GABA摄取。在5×10⁻⁸M [³H]GABA中孵育后，这些神经元出现大量标记，但在4℃孵育或无Na⁺离子存在时未检测到标记。二氨基丁酸（DABA）也完全阻断了神经元对GABA的摄取，而β-丙氨酸在相似浓度下无效。在体外培养6天时，50%的神经元存在Na⁺和温度依赖性的GABA摄取。此外，在这些神经元中，80%的摄取对DABA或β-丙氨酸不敏感，而其余20%则被DABA阻断但不被β-丙氨酸阻断。在神经元将GABA释放到培养基中的能力方面也发现了重要的发育变化。自发GABA释放（即在含有5mM K⁺的常规培养基中发生的释放）在体外培养3天时高于6天时。然而，将K⁺浓度提高到56mM在体外培养3天时影响最小，但在体外培养6天时诱导GABA释放增加2至3倍。这种K⁺诱导的释放似乎依赖于Ca²⁺，因为在10mM Co²⁺存在时它会大幅减少。通过将视网膜细胞悬液接种在粘附性差的基质上，生成了含有汇合单层非神经元扁平细胞的培养物。视网膜非神经元细胞在体外培养的前10天表现出一种高亲和力的GABA摄取机制，该机制依赖于Na⁺和温度，被DABA降低85%，但实际上不受β-丙氨酸影响。这种摄取机制在体外培养第二周结束时似乎丧失，在培养21天后无法检测到。