Transcriptional profiling of depolarization-dependent phenotypic alterations in primary cultures of developing granule neurons.
Transcriptional profiling of depolarization-dependent phenotypic alterations in primary cultures of developing granule neurons.
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发育中的颗粒神经元原代培养物中去极化依赖性表型改变的转录谱。
DOI：
10.1016/j.brainres.2006.08.043
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发表时间：
2006
期刊：
影响因子：
2.9
通讯作者：
Vallano,MaryLou
中科院分区：
文献类型：
--
作者：
Bui,CuongJ;McGann,AlexandraC;Middleton,FrankA;Beaman-Hall,CarolM;Vallano,MaryLou
大鼠小脑颗粒神经元培养在培养基中补充高氯化钾被广泛用作模型，以检查神经活动和钙依赖的基因表达之间的耦合。据信升高的（25 mM）KCl模拟内源性神经活动，因为它促进去极化和Ca+2依赖性存活以及成熟的某些方面。相比之下，在含有5 mM KCl的标准培养基中生长的颗粒神经元中，至少有一半在体外从24天开始发生凋亡。然而，越来越多的证据表明，慢性去极化诱导表型异常，而在含有5 mM KCl的化学成分确定的培养基中生长更接近于组成型表型。为了检验这一点，寡核苷酸微阵列和RT-PCR选择的mRNA被用来比较在5 mM和25 mM KCl中生长的培养物的转录谱。在某些情况下，还测试了N-甲基-d-天冬氨酸（NMDA），它与升高的KCl一样促进长期存活。观察到几个基因组的稳健变化，并表明在高KCl中生长：诱导通常观察不到的mRNA表达;抑制本应存在的mRNA表达;维持作为未成熟神经元标志物的mRNA表达。用NMDA代替高浓度KCl补充生长培养基也会产生类似的异常。总之，这些数据表明，在5 mM KCl中的生长更接近模拟颗粒神经元在体内的存活和成熟，因此应在未来的研究中采用。
Rat cerebellar granule neurons cultured in medium supplemented with elevated KCl are extensively used as a model to examine the coupling between neural activity and Ca2+-dependent gene expression. Elevated (25 mM) KCl is believed to mimic endogenous neural activity because it promotes depolarization and Ca+2-dependent survival and some aspects of maturation. By comparison, at least half of the granule neurons grown in standard medium containing 5 mM KCl undergo apoptosis beginning ∼4 days in vitro. However, accumulating evidence suggests that chronic depolarization induces phenotypic abnormalities whereas growth in chemically defined medium containing 5 mM KCl more closely resembles the constitutive phenotype. To examine this, oligonucleotide microarrays and RT-PCR of selected mRNAs were used to compare transcription profiles of cultures grown in 5 mM and 25 mM KCl. In some cases, N-methyl-d-aspartate (NMDA) which, like elevated KCl, promotes long-term survival was also tested. Robust changes in several gene groups were observed and indicated that growth in elevated KCl: induces expression of mRNAs that are not normally observed; represses expression of mRNAs that should be present; maintains expression of mRNAs that are markers of immature neurons. Supplementation of the growth medium with NMDA instead of elevated KCl produces similar abnormalities. Altogether, these data indicate that growth in 5 mM KCl more closely mimics survival and maturation of granule neurons in vivo and should therefore be adopted in future studies.