Absolute quantification of AMPA receptor subunit mRNAs in single hippocampal neurons

Abstract

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.