Absolute quantification of AMPA receptor subunit mRNAs in single hippocampal neurons

alpha -Amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subun it (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: P CR-amplified with the same efficiency as GluR1-4 mRNAs. The mean mRNA numbe rs harvested in vitro from pyramidal-like neurons on day 9 were 1150 +/- 32 4 molecules of GlwR1, 1080 +/- 273 molecules of GluR2, 100 +/- 20 molecules of GluR3, and 50 +/- 10 molecules of GluR4 (mean +/- SEM, n = 12). In a no n-pyramidal neuronal population that expresses AMPA receptors characterized by high Ca2+ permeability, the numbers of GluR1, GluR3 and GluR4 mRNA mole cules harvested per cell were 354 +/- 64, 25 +/- 17 and 168 +/- 36, respect ively (n = 8). The GluR2 mRNA was not detected in this cell type. The calcu lated ratio of AMPAR mRNA molecules per total mRNA molecules was 1/240 in p yramidal-like neurons (1/500 for GluR2), being in the range obtained with t otal RNA from rat forebrain and cerebellum (1/170 and 1/3801 respectively). Finally, our results indicated that the proportion of GluR1-4 mRNA located : in neurites reached similar to 60% in pyramidal-like neurons. However, we found no evidence of preferential subcellular distribution of a given subu nit.