Absolute quantification of AMPA receptor subunit mRNAs in single hippocampal neurons.
alpha-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 Ca(2+) 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 approximately 60% in pyramidal-like neurons. However, we found no evidence of preferential subcellular distribution of a given subunit.
Tsuzuki
K
K
Department of Physiology, Gunma University School of Medicine, Gunma, Japan. tsuzuki@med.gunma-u.ac.jp
Lambolez
B
B
Rossier
J
J
Ozawa
S
S
D000818:Animals; D002452:Cell Count; D002478:Cells, Cultured; D018507:Gene Expression Regulation, Developmental; D006624:Hippocampus / Q000166:cytology*; D009457:Neuroglia / Q000166:cytology; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D018408:Patch-Clamp Techniques; D018075:RNA, Complementary; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D018091:Receptors, AMPA / Q000235:genetics*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*
D018075:RNA, Complementary; D012333:RNA, Messenger; D018091:Receptors, AMPA; C104723:glutamate receptor ionotropic, AMPA 3; C104724:glutamate receptor ionotropic, AMPA 4; C104722:glutamate receptor ionotropic, AMPA 2