Zhong J, Russell S L, Pritchett D B, Molinoff P B, Williams K
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104-6084.
Mol Pharmacol. 1994 May;45(5):846-53.
The expression of mRNAs encoding subunits of the N-methyl-D-aspartate (NMDA) receptor was examined in cortical neurons maintained in primary culture. Cultures were prepared from embryonic day 17 rat neocortex. At this developmental age, levels of NR1, NR2A, NR2B, and NR2C mRNA were low or undetectable. Expression of NR1 mRNA increased progressively between days 1 and 21 in vitro. The amount of NR2A mRNA did not change between days 1 and 7 but increased between days 7 and 21. In contrast, levels of NR2B mRNA increased between days 1 and 7, with little further change after day 7. The level of NR2B mRNA was approximately 4-fold higher than that of NR2A mRNA in 21-day cultures. Using ligand binding assays, the proportion of NMDA receptors having a low affinity for ifenprodil was also found to increase over time in culture. The increase in the expression of receptors having a low affinity for ifenprodil and the increase in NR1 and NR2A mRNAs were reduced or prevented by maintaining cells in medium with a low concentration of serum. The results are consistent with the hypothesis that inclusion of the NR2A subunit in native NMDA receptors is responsible for their low affinity for ifenprodil. Splice variants of NR1 lacking the 5' (amino-terminal) insert were found to be the predominant forms of NR1 in cultured neurons. Variants containing the 5' insert represented only a small (< or = 5%) fraction of total NR1 mRNA, and their proportion was not altered as a function of time in culture. Time-dependent changes in the properties of NMDA receptors and in the expression of subunit mRNA occurring in cultured neurons are similar to changes observed in developing rat brain. Thus, the developmental sequence of NMDA receptor expression that occurs in vivo is partially retained in neurons maintained in vitro.
在原代培养的皮质神经元中检测了编码N-甲基-D-天冬氨酸（NMDA）受体亚基的mRNA的表达。培养物取自胚胎第17天的大鼠新皮质。在这个发育阶段，NR1、NR2A、NR2B和NR2C mRNA的水平很低或无法检测到。体外培养第1天到第21天期间，NR1 mRNA的表达逐渐增加。NR2A mRNA的量在第1天到第7天没有变化，但在第7天到第21天增加。相反，NR2B mRNA的水平在第1天到第7天增加，第7天后变化不大。在21天的培养物中，NR2B mRNA的水平比NR2A mRNA高约4倍。使用配体结合试验，还发现对ifenprodil亲和力低的NMDA受体比例在培养过程中随时间增加。通过将细胞维持在低浓度血清的培养基中，对ifenprodil亲和力低的受体表达增加以及NR1和NR2A mRNA的增加被减少或阻止。这些结果与以下假设一致，即天然NMDA受体中包含NR2A亚基是其对ifenprodil亲和力低的原因。在培养的神经元中，发现缺乏5'（氨基末端）插入片段的NR1剪接变体是NR1的主要形式。含有5'插入片段的变体仅占总NR1 mRNA的一小部分（≤5%），其比例不会随培养时间而改变。培养的神经元中NMDA受体特性和亚基mRNA表达的时间依赖性变化与发育中的大鼠脑中观察到的变化相似。因此，体内发生的NMDA受体表达的发育序列在体外培养的神经元中部分保留。
