Vallano M L, Lambolez B, Audinat E, Rossier J
Department of Pharmacology, State University of New York Health Sciences Center, Syracuse 13210, USA.
J Neurosci. 1996 Jan 15;16(2):631-9. doi: 10.1523/JNEUROSCI.16-02-00631.1996.
Reverse-transcription PCR assays were used to measure levels of NMDA receptor (NR) subunit mRNAs encoding splice variants of NR1 (NR1a, -exon 5; NR1b, +exon 5) and the major NR2 subunits (NR2A, NR2B, and NR2C) in dissociated cerebellar granule cell cultures. Cultures chronically exposed to 25 mM KCl or 100 microM NMDA/15 mM KCl, which promote survival by stimulating Ca2+ influx through voltage-sensitive Ca2+ channels or NRs, were compared with 5 mM KCl culture conditions, which results in limited cell survival attributable to a lower level of NR stimulation by ambient glutamate. In situ granule-cell maturation is associated with downregulation of NR2B and increases both of NR2A and NR2C and in the ratio of NR1b/NR1a mRNAs. In culture, 25 mM KCl or NMDA rapidly induced NR2A and downregulated NR2B, followed by gradual induction of NR2C. In 5 mM KCl, a similar, rapid increase in NR2A was observed, but disappearance of NR2B occurred over a longer time course. By 9-12 d in vitro in 5 mM KCl, the relative proportions of all three NR2 mRNAs in surviving cells were not significantly different from cells cultured in 25 mM KCl. NR1a mRNA predominated at every stage of culture in 25 mM KCl or NMDA, however, whereas gradual induction of the mature-form NR1b was observed in 5 mM KCl. Although using high potassium- or NMDA-containing media enhanced granule cell survival, it did not reproduce the pattern of expression of NR mRNAs observed in situ, whereas this pattern was observed in granule cells surviving in 5 mM KCl.
逆转录聚合酶链反应（Reverse-transcription PCR）检测用于测量解离的小脑颗粒细胞培养物中编码NR1剪接变体（NR1a，-外显子5；NR1b，+外显子5）和主要NR2亚基（NR2A、NR2B和NR2C）的NMDA受体（NR）亚基mRNA水平。将长期暴露于25 mM KCl或100 μM NMDA/15 mM KCl的培养物与5 mM KCl培养条件进行比较，前者通过刺激电压敏感性Ca2+通道或NRs使Ca2+内流来促进存活，而后者由于环境谷氨酸对NR的刺激水平较低导致细胞存活有限。原位颗粒细胞成熟与NR2B的下调以及NR2A和NR2C的增加以及NR1b/NR1a mRNA的比例增加有关。在培养中，25 mM KCl或NMDA迅速诱导NR2A并下调NR2B,随后逐渐诱导NR2C。在5 mM KCl中，观察到NR2A有类似的快速增加，但NR2B的消失发生在更长的时间过程中。在体外培养9 - 12天时，在5 mM KCl中存活细胞中所有三种NR2 mRNA的相对比例与在25 mM KCl中培养的细胞没有显著差异。然而，在25 mM KCl或NMDA中，NR1a mRNA在培养的每个阶段都占主导地位，而在5 mM KCl中观察到成熟形式的NR1b逐渐诱导。尽管使用含高钾或NMDA的培养基可提高颗粒细胞存活率，但它并未重现原位观察到的NR mRNA表达模式，而在5 mM KCl中存活的颗粒细胞中观察到了这种模式。
