Absolute quantification of AMPA receptor subunit mRNAs in single hippocampal neurons
Absolute quantification of AMPA receptor subunit mRNAs in single hippocampal neurons
复制标题
单个海马神经元中 AMPA 受体亚基 mRNA 的绝对定量
作者：
K. Tsuzuki;B. Lambolez;J. Rossier;S. Ozawa
在全细胞膜片钳记录后，使用内标 RNA 通过单细胞 RT-PCR 对大鼠海马培养物中单个神经元表达的 α-氨基-3-羟基-5-甲基异恶唑-4-丙酸酯 (AMPA) 受体亚基 (GluR1-4) mRNA 进行定量。内标 RNA 源自具有单核苷酸取代的 GluR2，以与 GluR1-4 mRNA 相同的效率进行反转录和 PCR 扩增。第9天从锥体样神经元体外收获的平均mRNA数量为1150±324个GluR1分子、1080±273个GluR2分子、100±20个GluR3分子和50±10个GluR4分子（平均值±SEM， n = 12）。在表达具有高 Ca2+ 通透性的 AMPA 受体的非锥体神经元群体中，每个细胞收获的 GluR1、GluR3 和 GluR4 mRNA 分子数量分别为 354 ± 64、25 ± 17 和 168 ± 36 (n = 8)。在此细胞类型中未检测到 GluR2 mRNA。锥体样神经元中 AMPAR mRNA 分子与总 mRNA 分子的计算比率为 1/240（GluR2 为 1/500），在大鼠前脑和小脑总 RNA 获得的范围内（分别为 1/170 和 1/380）。最后，我们的结果表明，在锥体样神经元中，位于神经突的 GluR1-4 mRNA 比例达到~60%。然而，我们没有发现给定亚基优先亚细胞分布的证据。
α‐Amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) receptor subunit (GluR1–4) mRNAs expressed by single neurons in rat hippocampal cultures were quantified by single‐cell RT‐PCR using an internal standard RNA after whole‐cell patch‐clamp recording. The internal standard RNA, derived from GluR2 with a single nucleotide substitution, was reverse‐transcribed and PCR‐amplified with the same efficiency as GluR1–4 mRNAs. The mean mRNA numbers harvested in vitro from pyramidal‐like neurons on day 9 were 1150 ± 324 molecules of GluR1, 1080 ± 273 molecules of GluR2, 100 ± 20 molecules of GluR3, and 50 ± 10 molecules of GluR4 (mean ± SEM, n = 12). In a non‐pyramidal neuronal population that expresses AMPA receptors characterized by high Ca2+ permeability, the numbers of GluR1, GluR3 and GluR4 mRNA molecules harvested per cell were 354 ± 64, 25 ± 17 and 168 ± 36, respectively (n = 8). The GluR2 mRNA was not detected in this cell type. The calculated ratio of AMPAR mRNA molecules per total mRNA molecules was 1/240 in pyramidal‐like neurons (1/500 for GluR2), being in the range obtained with total RNA from rat forebrain and cerebellum (1/170 and 1/380, respectively). Finally, our results indicated that the proportion of GluR1–4 mRNA located in neurites reached ∼60% in pyramidal‐like neurons. However, we found no evidence of preferential subcellular distribution of a given subunit.